Compounds and methods for diagnosis and immunotherapy of tuberculosis

ABSTRACT

Compounds and methods for diagnosing tuberculosis or for inducing protective immunity against tuberculosis are disclosed. The compounds provided include polypeptides that contain at least one immunogenic portion of one or more  Mycobacterium  proteins and DNA molecules encoding such polypeptides. Diagnostic kits containing such polypeptides or DNA sequences and a suitable detection reagent may be used for the detection of  Mycobacterium  infection in patients and biological samples. Antibodies directed against such polypeptides are also provided. In addition, such compounds may be formulated into vaccines and/or pharmaceutical compositions for immunization against  Mycobacterium  infection.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to patent application No.60/185,037, filed Feb. 25, 2000; and patent application No. 60/223,828,filed Aug. 8, 2000, herein each incorporated by reference in itsentirety.

The present application is related to U.S. patent application Ser. No.08/859,381, filed May 20, 1997 (abandoned); Ser. No. 08/858,998, filedMay 20, 1997 (abandoned); Ser. No. 09/073,010, filed May 5, 1998; andSer. No. 09/073,009, filed May 5, 1998; and to PCT application Nos.PCT/US98/10407, filed May 20, 1998; and PCT/US98/10514, filed May 20,1998, herein each incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

Tuberculosis is a chronic, infectious disease, that is generally causedby infection with Mycobacterium tuberculosis. It is a major disease indeveloping countries, as well as an increasing problem in developedareas of the world, with about 8 million new cases and 3 million deathseach year. Although the infection may be asymptomatic for a considerableperiod of time, the disease is most commonly manifested as an acuteinflammation of the lungs, resulting in fever and a nonproductive cough.If left untreated, serious complications and death typically result.

Although tuberculosis can generally be controlled using extendedantibiotic therapy, such treatment is not sufficient to prevent thespread of the disease. Infected individuals may be asymptomatic, butcontagious, for some time. In addition, although compliance with thetreatment regimen is critical, patient behavior is difficult to monitor.Some patients do not complete the course of treatment, which can lead toineffective treatment and the development of drug resistance.

Inhibiting the spread of tuberculosis will require effective vaccinationand accurate, early diagnosis of the disease. Currently, vaccinationwith live bacteria is the most efficient method for inducing protectiveimmunity. The most common Mycobacterium employed for this purpose isBacillus Calmette-Guerin (BCG), an avirulent strain of Mycobacteriumbovis. However, the safety and efficacy of BCG is a source ofcontroversy and some countries, such as the United States, do notvaccinate the general public. Diagnosis is commonly achieved using askin test, which involves intradermal exposure to tuberculin PPD(protein-purified derivative). Antigen-specific T cell responses resultin measurable induration at the injection site by 48-72 hours afterinjection, which indicates exposure to Mycobacterial antigens.Sensitivity and specificity have, however, been a problem with thistest, and individuals vaccinated with BCG cannot be distinguished frominfected individuals.

While macrophages have been shown to act as the principal effectors ofM. tuberculosis immunity, T cells are the predominant inducers of suchimmunity. The essential role of T cells in protection against M.tuberculosis infection is illustrated by the frequent occurrence of M.tuberculosis in AIDS patients, due to the depletion of CD4 T cellsassociated with human immunodeficiency virus (HIV) infection.Mycobacterium-reactive CD4 T cells have been shown to be potentproducers of gamma-interferon (IFN-γ), which, in turn, has been shown totrigger the anti-mycobacterial effects of macrophages in mice. While therole of IFN-γ in humans is less clear, studies have shown that1.25-dihydroxy-vitamin D3, either alone or in combination with IFN-γ ortumor necrosis factor-alpha, activates human macrophages to inhibit M.tuberculosis infection. Furthermore, it is known that IFN-γ stimulateshuman macrophages to make 1,25-dihydroxy-vitamin D3. Similarly, IL-12has been shown to play a role in stimulating resistance to M.tuberculosis infection. For a review of the immunology of M.tuberculosis infection, see Chan and Kaufmann, in Tuberculosis:Pathogenesis, Protection and Control, Bloom (ed.), ASM Press.Washington, D.C. (1994).

Accordingly, there is a need in the art for improved diagnostic methodsfor detecting tuberculosis, as well as for vaccines and methods forpreventing the infection. The present invention fulfills this need andfurther provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compounds and methods forpreventing and diagnosing tuberculosis.

In one embodiment, polypeptides are provided that comprise animmunogenic portion of a Mycobacterium antigen, preferably aMycobacterium tuberculosis antigen, or a variant of such an antigen thatdiffers only in conservative substitutions and/or modifications, whereinthe antigen comprises an amino acid sequence encoded by a polynucleotidehaving the nucleotide sequence recited in SEQ ID NO:145, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 162, and 164, thecomplements of said sequences, or a nucleotide sequence that hybridizesto the sequence set forth in SEQ ID NO:145, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 162, and 164, or an immunogenicfragment thereof. In a second embodiment, the present invention providespolypeptides comprising an immunogenic portion of a Mycobacteriumantigen, preferably a Mycobacterium tuberculosis antigen, having theamino acid sequence described in SEQ ID NO:146, 161, or 163 or variantsor immunogenic fragments thereof.

In related aspects, nucleotide sequences encoding the abovepolypeptides, recombinant expression vectors comprising these nucleotidesequences and host cells transformed or transfected with such expressionvectors are also provided. In particular, the present invention providesan isolated polynucleotide that specifically hybridizes under moderatelystringent conditions to a second polynucleotide comprising a nucleotidesequence selected from the group consisting of SEQ ID NO:145, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, and 164. In someembodiments, the isolated polynucleotide specifically hybridizes to thesecond polynucleotide under highly stringent conditions.

In another aspect, the present invention provides fusion proteinscomprising a first polypeptide encoded by a polynucleotide having thesequence set forth in SEQ ID NO:145, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 162, and 164, or a fragment thereof, and asecond polypeptide. In one embodiment, the first and second polypeptidesare heterologous. Alternatively, the fusion proteins of the inventionmay comprise a first polypeptide encoded by a polynucleotide having asequence selected from the group consisting of SEQ ID NO:145, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, and 164, or animmunogenic fragment thereof, and a known Mycobacterium antigen,preferably a M. tuberculosis antigen.

In further aspects of the subject invention, methods and diagnostic kitsare provided for detecting Mycobacterium infection in a patient. Themethods comprise contacting a biological sample with at least one of theabove polypeptides and detecting in the sample the presence ofantibodies that bind to the polypeptide or polypeptides, therebydetecting Mycobacterium infection in the biological sample. In apreferred embodiment, the Mycobacterium infection is a M. tuberculosisinfection.

Suitable biological samples include whole blood, sputum, serum, plasma,saliva, cerebrospinal fluid and urine. The diagnostic kits comprise oneor more of the above polypeptides in combination with a detectionreagent.

The present invention also provides methods for detecting Mycobacteriuminfection, comprising obtaining a biological sample from a patient,contacting the sample with at least one oligonucleotide primer in apolymerase chain reaction, the oligonucleotide primer being specific fora nucleotide sequence encoding the above polypeptides, and detecting inthe sample a nucleotide sequence that amplifies in the presence of thefirst and second oligonucleotide primers. In one embodiment, theoligonucleotide primer comprises at least about 10 contiguousnucleotides of such a nucleotide sequence. In a preferred embodiment,the Mycobacterium infection is a M. tuberculosis infection.

In a further aspect, the present invention provides a method fordetecting Mycobacterium infection in a patient, comprising obtaining abiological sample from the patient, contacting the sample with anoligonucleotide probe specific for a nucleotide sequence encoding theabove polypeptides, and detecting in the sample a nucleotide sequencethat hybridizes to the oligonucleotide probe. In one embodiment, theoligonucleotide probe comprises at least about 15 contiguous nucleotidesof such a nucleotide sequence. In a preferred embodiment, theMycobacterium infection is a M. tuberculosis infection.

In yet another aspect, methods are provided for detecting Mycobacteriuminfection in a patient, such methods comprising the steps of contactinga biological sample with a polypeptide, wherein the polypeptidecomprises an amino acid sequence encoded by a polynucleotide having anucleotide sequence selected from the group consisting of SEQ ID NO:145,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, and164, the complements of said sequences, or a nucleotide sequence thathybridizes to a sequence selected from the group consisting of SEQ IDNO:145, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162,and 164, or an immunogenic fragment thereof, and detecting in the samplethe presence of antibodies that bind to the polypeptide, therebydetecting Mycobacterium infection in the biological sample. In apreferred embodiment, the Mycobacterium infection is a M. tuberculosisinfection. Diagnostic kits for use in such methods are also provided.

In another aspect, the present invention provides antibodies, bothpolyclonal and monoclonal, that bind to the polypeptides describedabove, as well as methods for their use in the detection ofMycobacterium infection.

Within other aspects, the present invention provides pharmaceuticalcompositions that comprise one or more of the above polypeptides, or apolynucleotide encoding such polypeptides, and a physiologicallyacceptable carrier or an adjuvant, e.g., SBAS-2, QS-21, ENHANZYN(Detox), MPL, 3D-MPL, CWS, GM-CSF, SAF, ISCOMS, MF-59, RC-529, AS2,AS2′, AS2″, AS4, AS6, TDM, AGP, CPG, Leif, saponin, and saponinmimetics, and derivatives thereof or mixtures thereof. In anotheraspect, the present invention provides pharmaceutical compositions thatcomprise one or more of the above polypeptides, or a polynucleotideencoding such polypeptides, and an adjuvant such as BCG. In anotheraspect the present invention provides methods in which one or more ofthe above polypeptides, or a polynucleotide encoding such polypeptidesis administered to a subject who has been exposed to BCG. The inventionalso provides vaccines comprising one or more of the polypeptides asdescribed above and a non-specific immune response enhancer, togetherwith vaccines comprising one or more polynucleotides encoding suchpolypeptides and a non-specific immune response enhancer.

In yet another aspect, methods are provided for inducing protectiveimmunity in a patient, comprising administering to a patient aneffective amount of one or more of the above polypeptides.

In further aspects of this invention, methods and diagnostic kits areprovided for detecting tuberculosis in a patient. The methods comprisecontacting dermal cells of a patient with one or more of the abovepolypeptides and detecting an immune response on the patient's skin. Thediagnostic kits comprise one or more of the above polypeptides incombination with an apparatus sufficient to contact the polypeptide(s)with the dermal cells of a patient.

In yet another aspect, methods are provided for detecting tuberculosisin a patient, such methods comprising contacting dermal cells of apatient with one or more polypeptides encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO:145, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 162, and 164, the complements ofsaid sequences, or nucleotide sequences that hybridize to a sequenceselected from the group consisting of SEQ ID NO:145, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 162, and 164, and detecting animmune response on the patient's skin. Diagnostic kits for use in suchmethods are also provided.

In additional aspects of the invention, methods are provided forinhibiting the development of a Mycobacterium infection in a patient. Inone embodiment, inhibiting the development of a Mycobacterium infectioncomprises administering to a patient an effective amount of apharmaceutical composition or a vaccine of the invention. In anotherembodiment, inhibiting the development of a Mycobacterium infection inthe patient comprises administering to a patient an effective amount ofan antibody of the invention. In a preferred embodiment, theMycobacterium infection is a M. tuberculosis infection.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the stimulation of proliferation andinterferon-γ production, respectively, in T cells derived from a firstPPD-positive donor (referred to as D7) by recombinant ORF-2 andsynthetic peptides to ORF-2.

FIGS. 2A and 2B illustrate the stimulation of proliferation andinterferon-γ production, respectively, in T cells derived from a secondPPD-positive donor (referred to as D160) by recombinant ORF-2 andsynthetic peptides to ORF-2.

FIG. 3 shows the nucleotide sequence of mTTC#3 (SEQ ID NO:145).

FIG. 4 shows the amino acid sequence of mTCC#3 (SEQ ID NO:146).

FIG. 5 shows the 5′ nucleotide sequence of P1 (SEQ ID NO:149).

FIG. 6 shows the nucleotide sequence of P2 (SEQ ID NO:150).

FIG. 7 shows the 3′ nucleotide sequence of P3 (SEQ ID NO:151).

FIG. 8 shows the nucleotide sequence of P4 (SEQ ID NO:152).

FIG. 9 shows the nucleotide sequence of P6 (SEQ ID NO:153)

FIG. 10 shows the nucleotide sequence of P7 (SEQ ID NO:154)

FIG. 11 shows the nucleotide sequence of P8 (SEQ ID NO:155)

FIG. 12 shows the nucleotide sequence of P9 (SEQ ID NO:156)

FIG. 13 shows the 5′ nucleotide sequence of P10 (SEQ ID NO:157)

FIG. 14 shows the 5′ nucleotide sequence of P11 (SEQ ID NO:158)

FIG. 15 shows the 3′ nucleotide sequence of P12 (SEQ ID NO:159)

FIG. 16 shows the full length nucleotide and amino acid sequence of MO-1(SEQ ID NO:160 (nucleotide) and SEQ ID NO:161 (amino acid).

FIG. 17 shows the full length nucleotide and amino acid sequence of MO-2(SEQ ID NO:162 (nucleotide) and SEQ ID NO:163 (amino acid).

FIG. 18 shows the full length nucleotide sequence of TbH4/XP-1 (MTB48)(SEQ ID NO:164).

SEQ ID NO:1 is the cDNA sequence of Tb224

SEQ ID NO:2 is the cDNA sequence of Tb636

SEQ ID NO:3 is the cDNA sequence of Tb424

SEQ ID NO:4 is the cDNA sequence of Tb436

SEQ ID NO:5 is the cDNA sequence of Tb398

SEQ ID NO:6 is the cDNA sequence of Tb508

SEQ ID NO:7 is the cDNA sequence of Tb441

SEQ ID NO:8 is the cDNA sequence of Tb475

SEQ ID NO:9 is the cDNA sequence of Tb488

SEQ ID NO:10 is the cDNA sequence of Tb465

SEQ ID NO:11 is the cDNA sequence of Tb431

SEQ ID NO:12 is the cDNA sequence of Tb472 SEQ ID NO:13 is the predictedamino acid sequence of Tb224

SEQ ID NO:14 is the predicted amino acid sequence of Tb636

SEQ ID NO:15 is the predicted amino acid sequence of Tb431

SEQ ID NO:16 is the amino acid sequence of Tb424 ORF-1

SEQ ID NO:17 is the amino acid sequence of Tb424 ORF-2

SEQ ID NO:18 is the amino acid sequence of Tb436 ORF-1

SEQ ID NO:19 is the amino acid sequence of Tb436 ORF-2

SEQ ID NO:20 is the amino acid sequence of Tb398 ORF-1

SEQ ID NO:21 is the amino acid sequence of Tb398 ORF-2

SEQ ID NO:22 is the amino acid sequence of Tb508 ORF-1

SEQ ID NO:23 is the amino acid sequence of Tb508 ORF-2

SEQ ID NO:24 is the amino acid sequence of Tb441 ORF-1

SEQ ID NO:25 is the amino acid sequence of Tb441 ORF-2

SEQ ID NO:26 is the amino acid sequence of Tb475 ORF-1

SEQ ID NO:27 is the amino acid sequence of Tb475 ORF-2

SEQ ID NO:28 is the amino acid sequence of Tb488 ORF-1

SEQ ID NO:29 is the amino acid sequence of Tb488 ORF-2

SEQ ID NO:30 is the amino acid sequence of Tb465 ORF-1

SEQ ID NO:31 is the amino acid sequence of Tb465 ORF-2

SEQ ID NO:32 is the amino acid sequence of Tb424 ORF-U

SEQ ID NO:33 is the amino acid sequence of Tb436 ORF-U

SEQ ID NO:34 is the amino acid sequence of ORF-1-1

SEQ ID NO:35 is the amino acid sequence of ORF-1-2

SEQ ID NO:36 is the amino acid sequence of ORF-1-3

SEQ ID NO:37 is the amino acid sequence of ORF-1-4

SEQ ID NO:38 is the amino acid sequence of ORF-1-5

SEQ ID NO:39 is the amino acid sequence of ORF-1-6

SEQ ID NO:40 is the amino acid sequence of ORF-1-7

SEQ ID NO:41 is the amino acid sequence of ORF-1-8

SEQ ID NO:42 is the amino acid sequence of ORF-1-9

SEQ ID NO:43 is the amino acid sequence of ORF-1-10

SEQ ID NO:44 is the amino acid sequence of ORF-1-11

SEQ ID NO:45 is the amino acid sequence of ORF-1-12

SEQ ID NO:46 is the amino acid sequence of ORF-1-13

SEQ ID NO:47 is the amino acid sequence of ORF-1-14

SEQ ID NO:48 is the amino acid sequence of ORF-1-15

SEQ ID NO:49 is the amino acid sequence of ORF-1-16

SEQ ID NO:50 is the amino acid sequence of ORF-1-17

SEQ ID NO:51 is the amino acid sequence of ORF-2-1

SEQ ID NO:52 is the amino acid sequence of ORF-2-2

SEQ ID NO:53 is the amino acid sequence of ORF-2-3

SEQ ID NO:54 is the amino acid sequence of ORF-2-4

SEQ ID NO:55 is the amino acid sequence of ORF-2-5

SEQ ID NO:56 is the amino acid sequence of ORF-2-6

SEQ ID NO:57 is the amino acid sequence of ORF-2-7

SEQ ID NO:58 is the amino acid sequence of ORF-2-8

SEQ ID NO:59 is the amino acid sequence of ORF-2-9

SEQ ID NO:60 is the amino acid sequence of ORF-2-10

SEQ ID NO:61 is the amino acid sequence of ORF-2-11

SEQ ID NO:62 is the amino acid sequence of ORF-2-12

SEQ ID NO:63 is the amino acid sequence of ORF-2-13

SEQ ID NO:64 is the amino acid sequence of ORF-2-14

SEQ ID NO:65 is the amino acid sequence of ORF-2-15

SEQ ID NO:66 is the amino acid sequence of ORF-2-16

SEQ ID NO:67 is the amino acid sequence of ORF-2-17

SEQ ID NO:68 is the amino acid sequence of ORF-2-18

SEQ ID NO:69 is the amino acid sequence of ORF-2-19

SEQ ID NO:70 is the amino acid sequence of ORF-2-20

SEQ ID NO:71 is the amino acid sequence of ORF-2-21

SEQ ID NO:72 is the amino acid sequence of ORF-2-22

SEQ ID NO:73 is the amino acid sequence of ORF-2-23

SEQ ID NO:74 is the amino acid sequence of ORF-2-24

SEQ ID NO:75 is the amino acid sequence of ORF-2-25

SEQ ID NO:76 is the amino acid sequence of ORF-2-26

SEQ ID NO:77 is the amino acid sequence of ORF-2-27

SEQ ID NO:78 is the amino acid sequence of ORF-2-28

SEQ ID NO:79 is the amino acid sequence of ORF-2-29

SEQ ID NO:80 is the amino acid sequence of ORF-2-30

SEQ ID NO:81-82 are the amino acid sequence of two overlapping peptidesto the open reading frame of Tb224

SEQ ID NO:83 is the full-length cDNA sequence of Tb431 (which containsan ORF encoding Mtb-40)

SEQ ID NO:84 is the amino acid sequence of MSF-1

SEQ ID NO:85 is the amino acid sequence of MSF-2

SEQ ID NO:86 is the amino acid sequence of MSF-3

SEQ ID NO:87 is the amino acid sequence of MSF-4

SEQ ID NO:88 is the amino acid sequence of MSF-5

SEQ ID NO:89 is the amino acid sequence of MSF-6

SEQ ID NO:90 is the amino acid sequence of MSF-7

SEQ ID NO:91 is the amino acid sequence of MSF-8

SEQ ID NO:92 is the amino acid sequence of MSF-9

SEQ ID NO:93 is the amino acid sequence of MSF-10

SEQ ID NO:94 is the amino acid sequence of MSF-11

SEQ ID NO:95 is the amino acid sequence of MSF-12

SEQ ID NO:96 is the amino acid sequence of MSF-13

SEQ ID NO:97 is the amino acid sequence of MSF-14

SEQ ID NO:98 is the amino acid sequence of MSF-15

SEQ ID NO:99 is the amino acid sequence of MSF-16

SEQ ID NO:100 is the amino acid sequence of MSF-17

SEQ ID NO:101 is the amino acid sequence of MSF-18

SEQ ID NO:102 is the cDNA sequence of Tb867

SEQ ID NO:103 is the cDNA sequence of Tb391

SEQ ID NO:104 is the cDNA sequence of Tb470

SEQ ID NO:105 is the cDNA sequence of Tb838

SEQ ID NO:106-107 are the cDNA sequences of Tb962

SEQ ID NO:108 is the full-length cDNA sequence of Tb472

SEQ ID NO:109 is the predicted amino acid sequence of the proteinencoded by Tb472 (referred to as MSL)

SEQ ID NO:110 is the amino acid sequence of MSL-1

SEQ ID NO:111 is the amino acid sequence of MSL-2

SEQ ID NO:112 is the amino acid sequence of MSL-3

SEQ ID NO:113 is the amino acid sequence of MSL-4

SEQ ID NO:114 is the amino acid sequence of MSL-5

SEQ ID NO:115 is the amino acid sequence of MSL-6

SEQ ID NO:116 is the amino acid sequence of MSL-7

SEQ ID NO:117 is the amino acid sequence of MSL-8

SEQ ID NO:118 is the amino acid sequence of MSL-9

SEQ ID NO:119 is the amino acid sequence of MSL-10

SEQ ID NO:120 is the amino acid sequence of MSL-11

SEQ ID NO:121 is the amino acid sequence of MSL-12

SEQ ID NO:122 is the amino acid sequence of MSL-13

SEQ ID NO:123 is the amino acid sequence of MSL-14

SEQ ID NO:124 is the amino acid sequence of MSL-15

SEQ ID NO:125 is the DNA sequence of the full-length open reading frameof Tb470 (which encodes Mtb-40)

SEQ ID NO:126 is the determined amino acid sequence of Mtb-40

SEQ ID NO:127 is the cDNA sequence of Tb366

SEQ ID NO:128 is the cDNA sequence of Tb433

SEQ ID NO:129 is the cDNA sequence of Tb439

SEQ ID NO:130-131 are the cDNA sequences of Tb372

SEQ ID NO:132 is the cDNA sequence of Tb390R5C6

SEQ ID NO:133-134 are the cDNA sequences of Tb390R2C11

SEQ ID NO:135 is the 5′ cDNA sequence of Y1-26C1

SEQ ID NO:136 is the 5′ cDNA sequence of Y1-86C11

SEQ ID NO:137 is the full-length cDNA sequence of hTcc#1

SEQ ID NO:138 is the predicted amino acid sequence of hTcc#1

SEQ ID NO:139 is the cDNA sequence of mTCC#1

SEQ ID NO:140 is the cDNA sequence of mTCC#2

SEQ ID NO:141 is the predicted amino acid sequence of mTCC# 1

SEQ ID NO:142 is the predicted amino acid sequence of mTCC#2

SEQ ID NO:143 is the amino acid sequence of MTb9.8

SEQ ID NO:144 is the amino acid sequence of Tb#470

SEQ ID NO:145 is the full length nucleotide sequence of mTTC#3

SEQ ID NO:146 is the predicted amino acid sequence of mTTC#3

SEQ ID NO:147 and 148 are the sequences of primers used to amplify thefull-length coding sequence of mTTC#3

SEQ ID NO:149 is the 5′ nucleotide sequence of P1

SEQ ID NO:150 is the nucleotide sequence of P2

SEQ ID NO:151 is the 3′ nucleotide sequence of P3

SEQ ID NO:152 is the nucleotide sequence of P4

SEQ ID NO:153 is the nucleotide sequence of P6

SEQ ID NO:154 is the nucleotide sequence of P7

SEQ ID NO:155 is the nucleotide sequence of P8

SEQ ID NO:156 is the nucleotide sequence of P9

SEQ ID NO:157 is the 5′ nucleotide sequence of P10

SEQ ID NO:158 is the 5′ nucleotide sequence of P11

SEQ ID NO:159 is the 3′ nucleotide sequence of P12

SEQ ID NO:160 is the full length nucleotide sequence of MO-1

SEQ ID NO:161 is the full length amino acid sequence of MO-1.

SEQ ID NO:162 is the full length nucleotide sequence of MO-2

SEQ ID NO:163 is the full length amino acid sequence of MO-2

SEQ ID NO:164 is the full length nucleotide sequence of TbH4/XP-1(MTB48).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS I. Introduction

As noted above, the present invention is generally directed tocompositions and methods for preventing, treating and diagnosingtuberculosis. In particular, the present invention relates toMycobacterium antigens, optionally from a species such as M.tuberculosis, M. bovis, M. smegmatis, BCG, M. leprae, M. scrofulaceum,M. avium-intracellulare, M. marinum, M. ulcerans, M. kansasii, M.xenopi, M. szulgai, M. fortuium, or M. chelonei. In particular, theinvention relates to Mycobacterium polypeptides and immunogenicfragments thereof, polynucleotides that encode the polypeptides andimmunogenic fragments thereof, and methods of using such compositions inthe treatment, prevention and diagnosis of Mycobacterium infection. Inone embodiment of the invention, the polypeptides of the invention areused to diagnose tuberculosis. In another embodiment of the invention,the polypeptides of the invention are used to induce an immune responsein a patient in order to prevent Mycobacterium infection, and inparticular tuberculosis, or to reduce the probability of pathologicalresponses typical of Mycobacterium infection, and in particulartuberculosis, in a patient. In another embodiment of the invention, thepolynucleotides of the invention are used to produce DNA vaccines, orfor diagnostic purposes.

II. Definitions

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs). The term also encompasses ribonucleotidesincluding HnRNA molecules, which contain introns and correspond to a DNAmolecule in a one-to-one manner, and mRNA molecules, which do notcontain introns. Additional coding or non-coding sequences may, but neednot, be present within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Polynucleotide variants may containone or more substitutions, additions, deletions and/or insertions, asfurther described below, preferably such that the immunogenicity of theencoded polypeptide is not diminished relative to the nativepolypeptide. The effect on the immunogenicity of the encoded polypeptidemay generally be assessed as described herein. The term “variants” alsoencompasses interspecies homologs. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds. Thus, for instance, a polypeptidecomprising an immunogenic portion of an antigen may consist entirely ofthe immunogenic portion, or may contain additional sequences. Theadditional sequences may be derived from the native Mycobacteriumantigen or may be heterologous, and such sequences may (but need not) beimmunogenic.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The compositions and methods of this invention also encompass variantsof the above polypeptides. A polypeptide “variant,” as used herein, is apolypeptide that differs from the recited polypeptide only inconservative substitutions and/or modifications, such that thetherapeutic and/or immunogenic properties of the polypeptide areretained. Polypeptide variants preferably exhibit at least about 70%,more preferably at least about 90% and most preferably at least about95% identity to the identified polypeptides. For polypeptides withimmunoreactive properties, variants may, alternatively, be identified bymodifying the amino acid sequence of one of the above polypeptides, andevaluating the immunoreactivity of the modified polypeptide. Forpolypeptides useful for the generation of diagnostic binding agents, avariant may be identified by evaluating a modified polypeptide for theability to generate antibodies that detect the presence or absence ofMycobacterium infection, and in particular tuberculosis. Alternatively,variants of the claimed antigens that may be usefully employed in theinventive diagnostic methods may be identified by evaluating modifiedpolypeptides for their ability to detect antibodies present in the seraof Mycobacterium-infected patients. Such modified sequences may beprepared and tested using, for example, the representative proceduresdescribed herein.

A “conservative substitution” applies to both amino acid and nucleicacid sequences. With respect to particular nucleic acid sequences,conservative substitutions refers to changes in the nucleic acidsequence that result in nucleic acids encoding identical or essentiallyidentical amino acid sequences, or where the nucleic acid does notencode an amino acid sequence, to essentially identical sequences.Because of the degeneracy of the genetic code, a large number offunctionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that eachcodon in a nucleic acid (except AUG, which is ordinarily the only codonfor methionine, and TGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Accordingly, each silent variation of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservative substitution” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid andwhere the alteration has minimal influence on the immunogenicproperties, secondary structure and hydropathic nature of thepolypeptide. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

“Immunogenic,” as used herein, refers to the ability to elicit an immuneresponse (e.g., cellular or humoral) in a patient, such as a human,and/or in a biological sample (in vitro). In particular, antigens thatare immunogenic (and immunogenic portions or other variants of suchantigens) are recognized by a B-cell and/or a T-cell surface antigenreceptor. Antigens that are immunogenic (and immunogenic portions orother variants of such antigens) are capable of stimulating cellproliferation, interleukin-12 production and/or interferon-γ productionin biological samples comprising one or more cells selected from thegroup of T cells, NK cells, B cells and macrophages, where the cells arederived from an Mycobacterium-immune individual. Polypeptides comprisingat least an immunogenic portion of one or more Mycobacterium antigensmay generally be used to detect tuberculosis or to induce protectiveimmunity against tuberculosis in a patient.

“Fusion polypeptide” or “fusion protein” refers to a protein having atleast two heterologous polypeptides covalently linked, preferablyMycobacterium sp. polypeptides, either directly or via an amino acidlinker. The polypeptides forming the fusion protein are typically linkedC-terminus to N-terminus, although they can also be linked C-terminus toC-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. Thepolypeptides of the fusion protein can be in any order. This term alsorefers to conservatively modified variants, polymorphic variants,alleles, mutants, subsequences, and interspecies homologs of theantigens that make up the fusion protein. Mycobacterium tuberculosisantigens are described in Cole et al., Nature 393:537 (1998). Thecomplete sequence of the Mycobacterium tuberculosis genome can be foundat http://www.sanger.ac.uk and at http://www.pasteur.fr/mycdb/ (MycDB).

An adjuvant refers to the components in a vaccine or therapeuticcomposition that increase the specific immune response to the antigen(see, e.g., Edelman, AIDS Res. Hum Retroviruses 8:1409-1411 (1992)).Adjuvants induce immune responses of the Th1-type and Th-2 typeresponse. Th1-type cytokines (e.g., IFN-γ, IL-2, and IL-12) tend tofavor the induction of cell-mediated immune response to an administeredantigen, while Th-2 type cytokines (e.g., IL-4, IL-5, Il-6, IL-10 andTNF-β) tend to favor the induction of humoral immune responses.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For stringent hybridization, a positive signalis at least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions include: 50%formamide, 5×SSC and 1% SDS incubated at 42° C. or 5×SSC and 1% SDSincubated at 65° C., with a wash in 0.2×SSC and 0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cased, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately” stringent hybridization conditions include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 55%, 60%, 65%, 70%, 75%, or 80% identity, preferably 85%, 90%,95%, 96%, 97%, 98%, 99% or higher identity over a specified windowregion), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the complement of a testsequence. Preferably, the identity exists over a region that is at leastabout 25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, A model of evolutionary change in proteins—Matricesfor detecting distant relationships, In: Dayhoff (ed.) Atlas of ProteinSequence and Structure, National Biomedical Research Foundation,Washington D.C. Vol. 5, Suppl. 3, pp. 345-358 (1978); Hein, UnifiedApproach to Alignment and Phylogenes pp. 626-645 Methods in Enzymologyvol. 183, Academic Press, Inc., San Diego, Calif. (1990); Higgins andSharp, CABIOS 5:151-153 (1989); Myers and Muller, CABIOS 4:11-17 (1988);Robinson, Comb. Theor 11:105 (1971); Santou and Nes, Mol. Biol. Evol.4:406-425 (1987); Sneath and Sokal, Numerical Taxonomy—the Principlesand Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.(1973); Wilbur and Lipman, Proc. Natl. Acad. Sci. USA 80:726-730 (1983).

Alternatively, optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or bymanual alignment and visual inspection (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. (1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see, e.g., Fundamental Immunology (Paul ed.,3d ed. (1993)). While various antibody fragments are defined in terms ofthe digestion of an intact antibody, one of skill will appreciate thatsuch fragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990))

As used herein, an antibody, or antigen-binding fragment thereof, issaid to “specifically bind” to a polypeptide of interest if it reacts ata detectable level (within, for example, an ELISA) with the polypeptideof interest, and does not react detectably with unrelated proteins undersimilar conditions. As used herein, “binding” refers to a noncovalentassociation between two separate molecules such that a complex isformed. The ability to bind may be evaluated by, for example,determining a binding constant for the formation of the complex. Thebinding constant is the value obtained when the concentration of thecomplex is divided by the product of the component concentrations. Ingeneral, two compounds are said to “bind,” in the context of the presentinvention, when the binding constant for complex formation exceeds about10³ l/mol. The binding constant may be determined using methods wellknown in the art.

As used herein, a “biological sample” is any antibody-containing sampleobtained from a patient. Preferably, the sample is whole blood, sputum,serum, plasma, saliva, cerebrospinal fluid or urine. More preferably,the sample is a blood, serum or plasma sample obtained from a patient ora blood supply.

In the context of the present invention, a “patient” refers to anywarm-blooded animal, preferably a human. A patient may be afflicted witha disease, or may be free of detectable disease and/or infection.

III. Preparation of Mycobacterium Polypeptides and Nucleic Acids

In general, Mycobacterium antigens and DNA sequences encoding suchantigens may be prepared using any of a variety of procedures. Here andthroughout the specification, the Mycobacterium antigens are preferablyM. tuberculosis antigens.

A. Polynucleotides of the Invention

DNA sequences encoding antigens may be identified, for example, byscreening an appropriate Mycobacterium genomic or cDNA expressionlibrary with sera obtained from patients infected with Mycobacterium.Alternatively, sera from mice immunized with Mycobacterium antigens canbe used. In some embodiments, sera is obtained from mice immunized withblood or urine from syngeneic mice infected with Mycobacterium. Suchscreens may generally be performed using techniques well known to thoseof ordinary skill in the art, such as those described in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y. (1989).

DNA sequences encoding the antigens of the present invention may also beobtained by screening an appropriate Mycobacterium cDNA or genomic DNAlibrary for DNA sequences that hybridize to degenerate oligonucleotidesderived from partial amino acid sequences of isolated antigens.Degenerate oligonucleotide sequences for use in such a screen may bedesigned and synthesized, and the screen may be performed as described,for example, in Sambrook et al., supra, and references cited therein.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then screened byhybridizing filters containing denatured bacterial colonies (or lawnscontaining phage plaques) with the labeled probe (see Sambrook et al.,supra). Hybridizing colonies or plaques are selected and expanded, andthe DNA is isolated for further analysis. cDNA clones may be analyzed todetermine the amount of additional sequence by, for example, PCR using aprimer from the partial sequence and a primer from the vector.Restriction maps and partial sequences may be generated to identify oneor more overlapping clones. The complete sequence may then be determinedusing standard techniques, which may involve generating a series ofdeletion clones. The resulting overlapping sequences are then assembledinto a single contiguous sequence. A full length cDNA molecule can begenerated by ligating suitable fragments, using well known techniques.

Amplification techniques may also be employed, using the aboveoligonucleotides in methods well known in the art, to isolate a nucleicacid probe from a cDNA or genomic library. The library screen forobtaining a full length coding sequence from a partial cDNA sequence maythen be performed using the isolated probe. Within such techniques,amplification is generally performed via PCR. Any of a variety ofcommercially available kits may be used to perform the amplificationstep. Primers may be designed using, for example, software well known inthe art. Primers are preferably 22-30 nucleotides in length, have a GCcontent of at least 50% and anneal to the target sequence attemperatures of about 68° C. to 72° C. The amplified region may besequenced and overlapping sequences assembled into a contiguoussequence.

One such amplification technique is inverse PCR (see Triglia et al.,Nucl. Acids Res. 16:8186 (1988)), which uses restriction enzymes togenerate a fragment in the known region of the gene. The fragment isthen circularized by intramolecular ligation and used as a template forPCR with divergent primers derived from the known region. Within analternative approach, sequences adjacent to a partial sequence may beretrieved by amplification with a primer to a linker sequence and aprimer specific to a known region. The amplified sequences are typicallysubjected to a second round of amplification with the same linker primerand a second primer specific to the known region. A variation on thisprocedure, which employs two primers that initiate extension in oppositedirections from the known sequence, is described in WO 96/38591. Anothersuch technique is known as “rapid amplification of cDNA ends” or RACE.This technique involves the use of an internal primer and an externalprimer, which hybridizes to a polyA region or vector sequence, toidentify sequences that are 5′ and 3′ of a known sequence. Optionally,capture PCR (Lagerstrom et al., PCR Methods Applic. 1:111-119 (1991))and walking PCR (Parker et al., Nucl. Acids. Res. 19:3055-60 (1991)) canalso be used. Methods for amplification further include the ligase chainreaction (LCR; see, e.g., EP patent application publication 320, 308),the Qbeta Replicase method (see, e.g., PCT/US87/00880), the isothermalamplification method, the Strand Displacement Amplification (SDA), thecyclic probe reaction (CPR), the transcription-based amplificationsystems (TAS; see, e.g., PCT/US88/10315), as well as other methods knownto those of skill in the art (see, e.g., GB patent application No.2,202,328; PCT/US89/01025; and EP patent application publication No.329,822). Other methods employing amplification may also be employed toobtain a full length cDNA sequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous fill length sequence.

Polynucleotide variants may generally be prepared by any method known inthe art, including chemical synthesis by, for example, solid phasephosphoramidite chemical synthesis. Modifications in a polynucleotidesequence may also be introduced using standard mutagenesis techniques,such as oligonucleotide-directed site-specific mutagenesis (see Adelmanet al., DNA 2:183 (1983)). Alternatively, RNA molecules may be generatedby in vitro or in vivo transcription of DNA sequences encoding aMycobacterium polypeptide, or portion thereof, provided that the DNA isincorporated into a vector with a suitable RNA polymerase promoter (suchas T7 or SP6). Certain portions may be used to prepare an encodedpolypeptide, as described infra. In addition, or alternatively, aportion may be administered to a patient such that the encodedpolypeptide is generated in vivo (e.g., by transfectingantigen-presenting cells, such as dendritic cells, with a cDNA constructencoding a Mycobacterium polypeptide, and administering the transfectedcells to the patient).

A portion of a sequence complementary to a coding sequence (i.e., anantisense polynucleotide) may also be used as a probe or to modulategene expression. cDNA constructs that can be transcribed into antisenseRNA may also be introduced into cells or tissues to facilitate theproduction of antisense RNA. An antisense polynucleotide may be used, asdescribed herein, to inhibit expression of a Mycobacterium protein.Antisense technology can be used to control gene expression throughtriple-helix formation, which compromises the ability of the doublehelix to open sufficiently for the binding of polymerases, transcriptionfactors or regulatory molecules (see Gee et al., In Huber and Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y. (1994)). Alternatively, an antisense molecule may be designed tohybridize with a control region of a gene (e.g., promoter, enhancer ortranscription initiation site), and block transcription of the gene; orto block translation by inhibiting binding of a transcript to ribosomes.

A portion of a coding sequence or of a complementary sequence may alsobe designed as a probe or primer to detect gene expression. Probes maybe labeled with a variety of reporter groups, such as radionuclides andenzymes, and are preferably at least 10 nucleotides in length, morepreferably at least 20 nucleotides in length and still more preferablyat least 30 nucleotides in length. Primers, as noted above, arepreferably 22-30 nucleotides in length.

Any polynucleotide may be further modified to increase stability invivo. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends; the use ofphosphorothioate or 2′ O-methyl rather than phosphodiesterase linkagesin the backbone; and/or the inclusion of nontraditional bases such asinosine, queosine and wybutosine, as well as acetyl- methyl-, thio- andother modified forms of adenine, cytidine, guanine, thymine and uridine.

Nucleotide sequences as described herein may be joined to a variety ofother nucleotide sequences using established recombinant DNA techniques.For example, a polynucleotide may be cloned into any of a variety ofcloning vectors, including plasmids, phagemids, lambda phage derivativesand cosmids. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors and sequencing vectors. Ingeneral, a vector will contain an origin of replication functional in atleast one organism, convenient restriction endonuclease sites and one ormore selectable markers. Other elements will depend upon the desireduse, and will be apparent to those of ordinary skill in the art.

Within certain embodiments, polynucleotides may be formulated so as topermit entry into a cell of a mammal, and expression therein. Suchformulations are particularly useful for therapeutic purposes, asdescribed infra. Those of ordinary skill in the art will appreciate thatthere are many ways to achieve expression of a polynucleotide in atarget cell, and any suitable method may be employed. For example, apolynucleotide may be incorporated into a viral vector such as, but notlimited to, adenovirus, adeno-associated virus, retrovirus, or vacciniaor other pox virus (e.g., avian pox virus). The polynucleotides may alsobe administered as naked plasmid vectors. Techniques for incorporatingDNA into such vectors are well known to those of ordinary skill in theart. A retroviral vector may additionally transfer or incorporate a genefor a selectable marker (to aid in the identification or selection oftransduced cells) and/or a targeting moiety, such as a gene that encodesa ligand for a receptor on a specific target cell, to render the vectortarget specific. Targeting may also be accomplished using an antibody,by methods known to those of ordinary skill in the art.

Other formulations for therapeutic purposes include colloidal dispersionsystems, such as macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. A preferred colloidal systemfor use as a delivery vehicle in vitro and in vivo is a liposome (i.e.,an artificial membrane vesicle). The preparation and use of such systemsis well known in the art.

B. Polypeptides of the Invention

Within the context of the present invention, polypeptides may compriseat least an immunogenic portion of a Mycobacterium antigen, or a variantthereof, as described herein. As noted above, a Mycobacterium antigen isa protein that is expressed by cells infected with Mycobacterium. In apreferred embodiment the Mycobacterium antigen is a Mycobacteriumtuberculosis antigen. Proteins that are Mycobacterium antigens alsoreact detectably within an immunoassay (such as an ELISA) with antiserafrom a patient infected with Mycobacterium, and preferably with M.tuberculosis. Polypeptides as described herein may be of any length.Additional sequences derived from the native protein and/or heterologoussequences may be present, and such sequences may (but need not) possessfurther immunogenic or antigenic properties.

Genomic or cDNA libraries derived from Mycobacterium, and preferablyfrom M. tuberculosis, may be screened directly using peripheral bloodmononuclear cells (PBMCs) or T cell lines or clones derived from one ormore Mycobacterium-immune individuals. In a preferred embodiment, theMycobacterium-immune individuals are M. tuberculosis-immune individuals.Direct library screens may generally be performed by assaying pools ofexpressed recombinant proteins for the ability to induce proliferationand/or interferon-γ production in T cells derived from aMycobacterium-immune individual. Potential T cell antigens may be firstselected based on antibody reactivity, as described above. Purifiedantigens are then evaluated for their ability to elicit an appropriateimmune response (e.g., cellular) using, for example, the representativemethods described infra. Immunogenic antigens may then be partiallysequenced using techniques such as traditional Edman chemistry (seeEdman and Berg, Eur. J. Biochem. 80:116-132 (1967)).

Immunogenic antigens may also be produced recombinantly using a DNAsequence that encodes the antigen, which has been inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence, and expressed in an appropriate host. Methods which arewell known to those skilled in the art may be used to constructexpression vectors containing sequences encoding a polypeptide ofinterest and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in Sambrook et al., supra; and Ausubel et al., supra.

Polypeptides may comprise a signal (or leader) sequence at theN-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

Portions and other variants of Mycobacterium antigens may be generatedby synthetic or recombinant means. Synthetic polypeptides having fewerthan about 100 amino acids, and generally fewer than about 50 aminoacids, may be generated using techniques well known in the art. Forexample, such polypeptides may be synthesized using any of thecommercially available solid-phase techniques, such as the Merrifieldsolid-phase synthesis method, where amino acids are sequentially addedto a growing amino acid chain (see Merrifield, J. Am. Chem. Soc.85:2149-2146 (1963)). Equipment, for automated synthesis of polypeptidesis commercially available from suppliers such as Perkin Elmer/AppliedBioSystems Division, Inc., Foster City, Calif., and may be operatedaccording to the manufacturer's instructions. Variants of a nativeantigen may generally be prepared using standard mutagenesis techniques,such as oligonucleotide-directed site-specific mutagenesis. Sections ofthe DNA sequence may also be removed using standard techniques to permitpreparation of truncated polypeptides.

Recombinant polypeptides containing portions and/or variants of a nativeantigen may be readily prepared from a DNA sequence encoding thepolypeptide using a variety of techniques well known to those ofordinary skill in the art. For example, supernatants from suitablehost/vector systems which secrete recombinant protein into culture mediamay be first concentrated using a commercially available filter.Following concentration, the concentrate may be applied to a suitablepurification matrix such as an affinity matrix or an ion exchange resin.Finally, one or more reverse phase HPLC steps can be employed to furtherpurify a recombinant protein.

Any of a variety of expression vectors known to those of ordinary skillin the art may be employed to express recombinant polypeptides of thepresent invention. Expression may be achieved in any appropriate hostcell (e.g., prokaryotic, yeast and higher eukaryotic cell) that has beentransformed or transfected with an expression vector containing a DNAmolecule that encodes a recombinant polypeptide. Suitable expressionvector/host systems include, but are not limited to, microorganisms suchas bacteria transformed with recombinant bacteriophage, plasmid, orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors; insect cell systems infected with virus expression vectors(e.g., baculovirus); plant cell systems transformed with virusexpression vectors (e.g., cauliflower mosaic virus (CaMV); tobaccomosaic virus (TMV)) or with bacterial expression vectors (e.g., Ti orpBR322 plasmids); or animal cell systems. Examples of expression vectorsfor use in bacterial systems include, e.g., multifunctional E. colicloning and expression vectors such as BLUESCRIPT (Stratagene) and pINvectors (see Van Heeke and Schuster, J. Biol. Chem. 264:5503-5509(1989)). In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used (see, e.g., Ausubel et al., supra;and Grant et al., Methods Enzymol. 153:516-544 (1987)). In cases whereplant expression vectors are used, the expression of sequences encodingpolypeptides may be driven by any of a number of promoters, including,but not limited to, the 35S and 19S promoters of CaMV, the omega leadersequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)), as well asplant promoters such as the small subunit of RUBISCO or heat-shockpromoters (Coruzzi et al., EMBO J. 3:1671-1680 (1984); Broglie et al.,Science 224:838-843 (1984); and Winter et al., Results Probl. CellDiffer. 17:85-105 (1991)). A variety of expression vectors are alsoavailable for expression in insect systems. For example, suitablevectors for expression in Spodoptera frugiperda cells or in Trichoplusiainclude, but are not limited to, the Autographa californica nuclearpolyhedrosis virus (AcNPV). Furthermore, viral-based expression systemscan also be used to express the polypeptide(s) of interest in mammalianhost cells. Preferably, the host cells employed are E. coli, yeast ormammalian cell lines, such as COS or CHO. The DNA sequences expressed inthis manner may encode naturally occurring antigens, portions ofnaturally occurring antigens, or other variants thereof.

In general, regardless of the method of preparation, the polypeptidesdisclosed herein are prepared in substantially pure form. Preferably,the polypeptides are at least about 80% pure, more preferably at leastabout 90% pure and most preferably at least about 99% pure. For use inthe methods described herein, however, such substantially purepolypeptides may be combined.

In one embodiment, the subject invention discloses polypeptidescomprising at least an immunogenic portion of a M. tuberculosis antigen(or a variant of such an antigen) that comprises the amino acidsequences encoded by (a) the DNA sequence of SEQ ID NO:145, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, and 164; (b) thecomplement of such DNA sequence, or (c) a DNA sequence substantiallyhomologous to the sequence of (a) or (b). In a related embodiment, thepresent invention provides polypeptides comprising at least animmunogenic portion of a M. tuberculosis antigen having the amino acidsequence provided in SEQ ID NO:146, 161, or 163, and variants thereof.

The Mycobacterium antigens provided herein include variants that areencoded by DNA sequences which are substantially homologous to one ormore of DNA sequences specifically recited herein.

C. Fusion Polypeptides

In one embodiment, the present invention provides fusion proteinscomprising multiple polypeptides of the invention or, alternatively, apolypeptide of the present invention and a known Mycobacterium antigen,preferably a M. tuberculosis antigen. Examples of such knownMycobacterium antigens include, but are not limited to, e.g., 38 kDantigen described in Andersen and Hansen, Infect. Immun. 57:2481-2488(1989) (Genbank Accession No. M30046) and ESAT-6 previously identifiedin M. bovis (Accession No. U34848) and in M. tuberculosis (Sorensen etal., Infec. Immun. 63:1710-1717 (1995). Examples of suitableMycobacterium antigens are disclosed in U.S. patent application Ser.Nos. 09/056,556, 09/223,040 and 09/287,849, and in U.S. provisionalpatent application Nos. 60/158,338 and 60/158,425, herein eachincorporated by reference. Variants of such fusion proteins are alsoprovided.

The fusion proteins of the present invention may also include a fusionpartner which may, for example, assist in providing T helper epitopes(an immunological fusion partner), preferably T helper epitopesrecognized by humans, or assist in expressing the protein (an expressionenhancer) at higher yields than the native recombinant protein. Certainpreferred fusion partners are both immunological and expressionenhancing fusion partners. Examples of such proteins include tetanus,tuberculosis and hepatitis proteins (see, e.g., Stoute et al., New Engl.J. Med. 336:86-91, 1997). Other fusion partners may be selected so as toincrease the solubility of the protein or to enable the protein to betargeted to desired intracellular compartments. Still further fusionpartners include affinity tags, which facilitate purification of theprotein.

Within preferred embodiments, an immunological fusion partner is derivedfrom protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner are included on theN-terminus to provide the polypeptide with additional exogenous T-cellepitopes and to increase the expression level in E. coli (thusfunctioning as an expression enhancer). The lipid tail ensures optimalpresentation of the antigen to antigen presenting cells. Other fusionpartners include the non-structural protein from influenzae virus, NS1(hemaglutinin). Typically, the N-terminal 81 amino acids are used,although different fragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292 (1986)). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798 (1992)). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

The fusion proteins of the present invention may also include a linkerpeptide between the first and second polypeptides. A peptide linkersequence may be employed to separate, for example, the first and secondpolypeptide components by a distance sufficient to ensure that eachpolypeptide folds into its secondary and tertiary structures. Such apeptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262 (1986); U.S. Pat. Nos. 4,935,233 and 4,751,180. The linkersequence may generally be from 1 to about 50 amino acids in length.Linker sequences are not required when the first and second polypeptideshave non-essential N-terminal amino acid regions that can be used toseparate the functional domains and prevent steric interference.

Fusion proteins may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion protein isexpressed as a recombinant protein in an expression system. Briefly, DNAsequences encoding the polypeptide components may be assembledseparately, and ligated into an appropriate expression vector. The 3′end of the DNA sequence encoding one polypeptide component is ligated,with or without a peptide linker, to the 5′ end of a DNA sequenceencoding the second polypeptide component so that the reading frames ofthe sequences are in phase. This permits translation into a singlefusion protein that retains the biological activity of both componentpolypeptides.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptide. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

In general, polypeptides (including fusion proteins) and polynucleotidesas described herein are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

D. Immunogenicity of the Polypeptides of the Invention

Regardless of the method of preparation, the antigens and immunogenicportions thereof described herein have the ability to induce animmunogenic response. More specifically, the antigens have the abilityto react with sera obtained from a Mycobacterium-infected individualand/or to induce proliferation and/or cytokine production (i.e.,interferon-γ and/or interleukin-12 production) in T cells, NK cells, Bcells and/or macrophages derived from a Mycobacterium-immune individual.Here and throughout the specification, the Mycobacterium-immuneindividual is preferably an M. tuberculosis-immune individual.

Reactivity with sera obtained from a Mycobacterium-infected individualmay be evaluated using, for example, the representative ELISA assaysdescribed herein, where an absorbance reading with sera from infectedindividuals that is at least three standard deviations above theabsorbance obtained with sera from uninfected individuals is consideredpositive.

The selection of cell type for use in evaluating an immunogenic responseto a antigen will, of course, depend on the desired response. Forexample, interleukin-12 production is most readily evaluated usingpreparations containing B cells and/or macrophages. AMycobacterium-immune individual (e.g., an M. tuberculosis-immuneindividual) is one who is considered to be resistant to the developmentof the disease (e.g., tuberculosis) by virtue of having mounted aneffective T cell response to Mycobacterium (i.e., substantially free ofdisease symptoms). Such individuals may be identified based on astrongly positive (i.e., greater than about 10 mm diameter induration)intradermal skin test response to tuberculosis proteins (PPD) and anabsence of any signs or symptoms of, e.g., tuberculosis disease. Tcells, NK cells, B cells and macrophages derived fromMycobacterium-immune individuals may be prepared using methods known tothose of ordinary skill in the art. For example, a preparation of PBMCs(i.e., peripheral blood mononuclear cells) may be employed withoutfurther separation of component cells. PBMCs may generally be prepared,for example, using density centrifugation through Ficoll™ (WinthropLaboratories. NY).

T cells for use in the assays described herein may also be purifieddirectly from PBMCs. Alternatively, an enriched T cell line reactiveagainst mycobacterial proteins, or T cell clones reactive to individualmycobacterial proteins, may be employed. Such T cell clones may begenerated by, for example, culturing PBMCs from Mycobacterium-immuneindividuals with mycobacterial proteins for a period of 2-4 weeks. Thisallows expansion of only the mycobacterial protein-specific T cells,resulting in a line composed solely of such cells. These cells may thenbe cloned and tested with individual proteins, using methods known tothose of ordinary skill in the art, to more accurately define individualT cell specificity. In general, antigens that test positive in assaysfor proliferation and/or cytokine production (i.e., interferon-γ and/orinterleukin-12 production) performed using T cells, NK cells, B cellsand/or macrophages derived from an Mycobacterium-immune individual areconsidered immunogenic. Such assays may be performed, for example, usingthe representative procedures described infra. Immunogenic portions ofsuch antigens may be identified using similar assays, and may be presentwithin the polypeptides described herein.

The ability of a polypeptide (e.g., an immunogenic antigen, or a portionor other variant thereof) to induce cell proliferation is evaluated bycontacting the cells (e.g., T cells and/or NK cells) with thepolypeptide and measuring the proliferation of the cells. In general,the amount of polypeptide that is sufficient for evaluation of about 10⁵cells ranges from about 10 ng/ml to about 100 μg/ml and preferably isabout 10 μg/ml. The incubation of a polypeptide with cells is typicallyperformed at 37° C. for about six days. Following incubation with thepolypeptide, the cells are assayed for a proliferative response, whichmay be evaluated by methods known to those of ordinary skill in the art,such as exposing the cells to a pulse of radiolabeled thymidine andmeasuring the incorporation of label into cellular DNA. In general, apolypeptide that results in at least a three fold increase inproliferation above background (i.e., the proliferation observed forcells cultured without polypeptide) is considered to be able to induceproliferation.

The ability of a polypeptide to stimulate the production of interferon-γand/or interleukin-12 in cells may be evaluated by contacting the cellswith the polypeptide and measuring the level of interferon-γ orinterleukin-12 produced by the cells. In general, the amount ofpolypeptide that is sufficient for the evaluation of about 10⁵ cellsranges from about 10 ng/ml to about 100 μg/ml and preferably is about 10μg/ml. The polypeptide may, but need not, be immobilized on a solidsupport, such as a bead or a biodegradable microsphere, such as thosedescribed in, e.g., U.S. Pat. Nos. 4,897,268 and 5,075,109. Theincubation of a polypeptide with the cells is typically performed at 37°C. for about six days. Following incubation with the polypeptide, thecells are assayed for interferon-γ and/or interleukin-12 (or one or moresubunits thereof) production, which may be evaluated by methods known tothose of ordinary skill in the art, such as an enzyme-linkedimmunosorbent assay (ELISA) or, in the case of the IL-12 P70heterodimer, a bioassay such as an assay measuring proliferation of Tcells. In general, a polypeptide that results in the production of atleast 50 pg of interferon-γ per ml of cultured supernatant (containing10⁴-10⁵ T cells per ml) is considered able to stimulate the productionof interferon-γ. A polypeptide that stimulates the production of atleast 10 pg/ml of IL-12 P70 subunit, and/or at least 100 pg/ml of IL-12P40 subunit, per 10⁵ macrophages or B cells (or per 3×10⁵ PBMC) isconsidered able to stimulate the production of IL-12.

In general, immunogenic antigens are those antigens that stimulateproliferation and/or cytokine production (i.e., interferon-γ and/orinterleukin-12 production) in T cells, NK cells, B cells and/ormacrophages derived from at least about 25% of Mycobacterium-immuneindividuals. Among these immunogenic antigens, polypeptides havingsuperior therapeutic properties may be distinguished based on themagnitude of the responses in the above assays and based on thepercentage of individuals for which a response is observed. In addition,antigens having superior therapeutic properties will not stimulateproliferation and/or cytokine production in vitro in cells derived frommore than about 25% of individuals that are not Mycobacterium-immune,thereby eliminating responses that are not specifically due toMycobacterium-responsive cells. Those antigens that induce a response ina high percentage of T cell, NK cell, B cell and/or macrophagepreparations from Mycobacterium-immune individuals (with a low incidenceof responses in cell preparations from other individuals) have superiortherapeutic properties.

Antigens with superior therapeutic properties may also be identifiedbased on their ability to diminish the severity of Mycobacteriuminfection in experimental animals, when administered as a vaccine.Suitable vaccine preparations for use on experimental animals aredescribed in detail below. Efficacy may be determined based on theability of the antigen to provide at least about a 50% reduction inbacterial numbers and/or at least about a 40% decrease in mortalityfollowing experimental infection. Suitable experimental animals include,e.g., mice, guinea pigs and primates.

Antigens having superior diagnostic properties may generally beidentified based on the ability to elicit a response in an intradermalskin test performed on an individual with active tuberculosis, but notin a test performed on an individual who is not infected withMycobacterium. Skin tests may generally be performed as described below,with a response of at least 5 mm induration considered positive.

Immunogenic portions of Mycobacterium antigens may be prepared andidentified using well known techniques, such as those summarized inPaul, Fundamental Immunology, 3d ed., Raven Press, pp. 243-247 (1993)and references cited therein. Such techniques include screeningpolypeptide portions of the native antigen for immunogenic propertiesand in particular, e.g., ability to react with antigen-specificantibodies, antisera and/or T-cell lines or clones. As used herein,antisera and antibodies are “antigen-specific” if they specifically bindto an antigen (i.e., they react with the protein in an ELISA or otherimmunoassay, and do not react detectably with unrelated proteins). Suchantisera and antibodies may be prepared as described herein, and usingwell known techniques. The representative ELISAs as well as theproliferation and cytokine production assays described herein maygenerally be employed in these screens. An immunogenic portion of apolypeptide is a portion that, within such representative assays,generates a signal or an immune response (e.g., proliferation,interferon-γ production and/or interleukin-12 production) that is notsubstantially less than that generated by the full length polypeptide.In other words, an immunogenic portion of a Mycobacterium antigengenerates at least about 20%, and preferably about 100%, of the signaland/or immune response induced by the full length antigen in the modelELISA or proliferation assay described herein, respectively. Animmunogenic portion may also, or alternatively, stimulate the productionof at least about 20%, and preferably about 100%, of the interferon-γand/or interleukin-12 induced by the full length antigen in the modelassay described herein. Such immunogenic portions may also react withinsuch assays at a level that is greater than the reactivity of the fulllength polypeptide. Such screens may generally be performed usingmethods well known to those of ordinary skill in the art, such as thosedescribed in Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory (1988). For use in the methods describedherein, substantially pure polypeptides may be combined.

IV. Antibodies

The present invention further provides agents, such as antibodies andantigen-binding fragments thereof, that specifically bind to thepolypeptides of the invention. Binding agents may be capable ofdifferentiating between patients infected or not with Mycobacterium, andin particular with M. tuberculosis, using the representative assaysprovided infra. In other words, antibodies or other binding agents thatbind to a Mycobacterium antigen will generate a signal indicating thepresence of tuberculosis in at least about 20% of patients with thedisease, and will generate a negative signal indicating the absence ofthe disease in at least about 90% of individuals without tuberculosis.To determine whether a binding agent satisfies this requirement,biological samples (e.g., blood, sera, urine, sputum, saliva, etc.) frompatients with and without tuberculosis (as determined using standardclinical tests) may be assayed as described herein for the presence ofpolypeptides that bind to the binding agent. It will be apparent that astatistically significant number of samples with and without the diseaseshould be assayed. Each binding agent should satisfy the above criteria;however, those of ordinary skill in the art will recognize that bindingagents may be used in combination to improve sensitivity.

Any agent that satisfies the above requirements may be a binding agent.For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof.

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art (see, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)).In general, antibodies can be produced by cell culture techniques,including the generation of monoclonal antibodies as described herein,or via transfection of antibody genes into suitable bacterial ormammalian cell hosts, in order to allow for the production ofrecombinant antibodies. In one technique, an immunogen comprising theimmunogenic polypeptide is initially injected into any of a wide varietyof mammals (e.g., mice, rats, rabbits, sheep and goats). In this step,the polypeptides of the invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Polyclonal antibodies raised to a fusion protein of the invention canalso be obtained by selecting only those polyclonal antibodies that arespecifically immunoreactive with the fusion protein of interest and notwith the individual polypeptide components of the fusion protein. Thisselection may be achieved by subtracting out antibodies that cross-reactwith the individual polypeptide components of the fusion protein ofinterest.

Alternatively, antibodies that recognize each or all of the individualpolypeptide components of a fusion protein may be useful in the contextof the present invention.

Monoclonal antibodies specific for the immunogenic polypeptide ofinterest may be prepared, for example, using the technique of Kohier andMilstein, Eur. J. Immunol. 6:511-519 (1976), and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as, e.g., a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (see Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory (1988)) and digested by papain to yield Fab andFc fragments. The Fab and Fc fragments may be separated by affinitychromatography on protein A bead columns.

Antibodies may be used in diagnostic tests to detect the presence ofMycobacterium antigens using assays similar to those detailed infra andother techniques well known to those of skill in the art, therebyproviding a methods for detecting Mycobacterium infection, and inparticular tuberculosis, in a patient.

Monoclonal antibodies of the present invention may be coupled to one ormore therapeutic agents. Suitable agents in this regard include, but arenot limited to, drugs, toxins, and derivatives thereof. Preferred drugsinclude, e.g., penicillin, rifampin, isoniazid, pyrazinamide,ethambutol, streptomycin, etc. These drugs can be obtained from anatural source or be semisynthetic or synthetic compounds. Preferredtoxins include ricin, abrin, Diphtheria toxin, cholera toxin, gelonin,Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, including, e.g., U.S.Pat. No. 4,671,958.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710), by irradiation of aphotolabile bond (e.g., U.S. Pat. No. 4,625,014), by hydrolysis ofderivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045), byserum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958),and acid-catalyzed hydrolysis (e.g., U.S. Pat. No. 4,569,789).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group. Suitable carriers includeproteins such as, e.g., albumins (e.g., U.S. Pat. No. 4,507,234),peptides and polysaccharides such as, e.g., aminodextran (e.g., U.S.Pat. No. 4,699,784). A carrier may also bear an agent by noncovalentbonding or by encapsulation, such as within a liposome vesicle (e.g.U.S. Pat. Nos. 4,429,008 and 4,873,088).

A variety of routes of administration for the antibodies andimmunoconjugates may be used. Typically, administration will be, e.g.,intravenous, intramuscular, subcutaneous, intranasal, or buccal. It willbe evident that the precise dose of the antibody/immunoconjugate willvary depending upon the antibody used, the antigen density in the cells,and the rate of clearance of the antibody.

V. T Cells

Immunotherapeutic compositions may also, or alternatively, comprise Tcells specific for a Mycobacterium antigen. Such cells may generally beprepared in vitro or ex vivo, using standard procedures. For example, Tcells may be isolated from bone marrow, peripheral blood or a fractionof bone marrow or peripheral blood of a patient, using a commerciallyavailable cell separation system, such as the CEPRATE™ system, availablefrom CellPro Inc., Bothell Wash. (see also U.S. Pat. Nos. 5,240,856 and5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, Tcells may be derived from related or unrelated humans, non-humanmammals, cell lines or cultures.

T cells may be stimulated with a Mycobacterium polypeptide, apolynucleotide encoding a Mycobacterium polypeptide and/or an antigenpresenting cell (APC) that expresses such a polypeptide. Suchstimulation is performed under conditions and for a time sufficient topermit the generation of T cells that are specific for the polypeptide.Preferably, a Mycobacterium polypeptide or polynucleotide is presentwithin a delivery vehicle, such as a microsphere, to facilitate thegeneration of specific T cells.

T cells are considered to be specific for a Mycobacterium polypeptide ifthe T cells kill target cells coated with the polypeptide or expressinga gene encoding the polypeptide. T cell specificity may be evaluatedusing any of a variety of standard techniques. For example, within achromium release assay or proliferation assay, a stimulation index ofmore than two fold increase in lysis and/or proliferation, compared tonegative controls, indicates T cell specificity. Such assays may beperformed, for example, as described in Chen et al., Cancer Res.54:1065-1070 (1994). Alternatively, detection of the proliferation of Tcells may be accomplished by a variety of known techniques. For example,T cell proliferation can be detected by measuring an increased rate ofDNA synthesis (e.g., by pulse-labeling cultures of T cells withtritiated thymidine and measuring the amount of tritiated thymidineincorporated into DNA). Contact with a Mycobacterium polypeptide (100ng/ml-100 μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days shouldresult in at least a two fold increase in proliferation of the T cells.Contact as described above for 2-3 hours should result in activation ofthe T cells, as measured using standard cytokine assays in which a twofold increase in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1, Wiley Interscience, Greene (1998)). T cells thathave been activated in response to a Mycobacterium polypeptide,polynucleotide or polypeptide-expressing APC may be CD4⁺ and/or CD8⁺ .Mycobacterium polypeptide-specific T cells may be expanded usingstandard techniques. Within preferred embodiments, the T cells arederived from a patient, or from a related or unrelated donor, and areadministered to the patient following stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to a Mycobacterium polypeptide, polynucleotide or APC can beexpanded in number either in vitro or in vivo. Proliferation of such Tcells in vitro may be accomplished in a variety of ways. For example,the T cells can be re-exposed to a Mycobacterium polypeptide (e.g., ashort peptide corresponding to an immunogenic portion of such apolypeptide) with or without the addition of T cell growth factors, suchas interleukin-2, and/or stimulator cells that synthesize aMycobacterium polypeptide. Alternatively, one or more T cells thatproliferate in the presence of a Mycobacterium polypeptide can beexpanded in number by cloning. Methods for cloning cells are well knownin the art, and include limiting dilution. Following expansion, thecells may be administered back to the patient as described, for example,by Chang et al., Crit. Rev. Oncol. Hematol. 22:213 (1996).

VI. Diagnostic Assays

A. Diagnostic assays with Mycobacterium polypeptides

In another aspect, the present invention provides methods for using thepolypeptides described above to diagnose Mycobacterium infection, and inparticular tuberculosis. In this aspect, methods are provided fordetecting Mycobacterium infection in a biological sample, using one ormore of the above polypeptides, alone or in combination. In embodimentsin which multiple polypeptides are employed, polypeptides other thanthose specifically described herein, such as the 38 kD antigen describedabove, may be included. The polypeptide(s) are used in an assay, asdescribed infra, to determine the presence or absence of antibodies tothe polypeptide(s) in a biological sample (e.g., whole blood, sputum,serum, plasma, saliva, cerebrospinal fluid, urine, etc.) relative to apredetermined cut-off value. The presence of such antibodies indicatesprevious sensitization to mycobacterial antigens which may be indicativeof Mycobacterium infection, and in particular tuberculosis.

In embodiments in which more than one polypeptide is employed, thepolypeptides used are preferably complementary (i.e., one componentpolypeptide will tend to detect infection in samples where the infectionwould not be detected by another component polypeptide). Complementarypolypeptides may generally be identified by using each polypeptideindividually to evaluate serum samples obtained from a series ofpatients known to be infected with Mycobacterium. After determiningwhich samples test positive (as described below) with each polypeptide,combinations of two or more polypeptides may be formulated that arecapable of detecting infection in most, or all, of the samples tested.Such polypeptides are complementary. For example, approximately 25-30%of sera from tuberculosis-infected individuals are negative forantibodies to any single protein, such as the above-mentioned 38 kDantigen. Complementary polypeptides may, therefore, be used incombination with the 38 kD antigen to improve sensitivity of adiagnostic test.

There are a variety of assay formats known to those of ordinary skill inthe art for using one or more polypeptides to detect antibodies in asample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory (1988), which is incorporated herein byreference. In general, the presence or absence of tuberculosis in apatient may be determined by (a) contacting a biological sample obtainedfrom a patient with one or more polypeptides or fusion proteins of theinvention; (b) detecting in the sample a level of antibody that binds tothe polypeptide(s) or the fusion protein(s); and (c) comparing the levelof antibody with a predetermined cut-off value.

In a preferred embodiment, the assay involves the use of a polypeptideimmobilized on a solid support to bind to and remove the antibody fromthe sample. The bound antibody may then be detected using a detectionreagent that contains a reporter group. Suitable detection reagentsinclude antibodies that bind to the antibody/polypeptide complex andfree polypeptide labeled with a reporter group (e.g., in asemi-competitive assay). Alternatively, a competitive assay may beutilized, in which an antibody that binds to the polypeptide of interestis labeled with a reporter group and allowed to bind to the immobilizedantigen after incubation of the antigen with the sample. The extent towhich components of the sample inhibit the binding of the labeledantibody to the polypeptide is indicative of the reactivity of thesample with the immobilized polypeptide.

The solid support may be any solid material known to those of ordinaryskill in the art to which the antigen may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681.

The polypeptides may be bound to the solid support using a variety oftechniques known to those of ordinary skill in the art, which are amplydescribed in the patent and scientific literature. In the context of thepresent invention, the term “bound” refers to both noncovalentassociation, such as adsorption, and covalent attachment (which may be adirect linkage between the antigen and functional groups on the supportor may be a linkage by way of a cross-linking agent). Binding byadsorption to a well in a microtiter plate or to a membrane ispreferred. In such cases, adsorption may be achieved by contacting thepolypeptide, in a suitable buffer, with the solid support for a suitableamount of time. The contact time varies with temperature, but istypically between about 1 hour and 1 day. In general, contacting a wellof a plastic microtiter plate (such as polystyrene or polyvinylchloride)with an amount of polypeptide ranging from about 10 ng to about 1 μg,and preferably about 100 ng, is sufficient to bind an adequate amount ofantigen.

Covalent attachment of the polypeptide of interest to a solid supportmay generally be achieved by first reacting the support with abifunctional reagent that reacts with both the support and a functionalgroup, such as a hydroxyl or amino group, on the polypeptide. Forexample, the polypeptide may be bound to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on thepolypeptide (see, e.g., Pierce Immunotechnology Catalog and Handbook, atA12-A13 (1991)).

In certain embodiments, the assay is an enzyme linked immunosorbentassay (ELISA). This assay may be performed by first contacting apolypeptide antigen that has been immobilized on a solid support,commonly the well of a microtiter plate, with the sample, such thatantibodies present within the sample that recognize the polypeptide ofinterest are allowed to bind to the immobilized polypeptide. Unboundsample is then removed from the immobilized polypeptide and a detectionreagent capable of binding to the immobilized antibody-polypeptidecomplex is added. The amount of detection reagent that remains bound tothe solid support is then determined using a method appropriate for thespecific detection reagent.

More specifically, once the polypeptide is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or TWEEN 20™(Sigma Chemical Co., St. Louis, Mo.), may be employed. The immobilizedpolypeptide is then incubated with the sample, and the antibody isallowed to bind to the antigen. The sample may be diluted with asuitable diluent, such as phosphate-buffered saline (PBS) prior toincubation. In general, an appropriate contact time (i.e., incubationtime) is a period of time that is sufficient to detect the presence ofantibody within a Mycobacterium-infected sample. Preferably, the contacttime is sufficient to achieve a level of binding that is at least 95% ofthat achieved at equilibrium between bound and unbound antibody. Thoseof ordinary skill in the art will recognize that the time necessary toachieve equilibrium may be readily determined by assaying the level ofbinding that occurs over a period of time. At room temperature, anincubation time of about 30 minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% TWEEN 20™. Detectionreagent may then be added to the solid support. An appropriate detectionreagent is any compound that binds to the immobilizedantibody-polypeptide complex and that can be detected by any of avariety of means known to those in the art. Preferably, the detectionreagent contains a binding agent (such as, for example, Protein A,Protein G, immunoglobulin, lectin or free antigen) conjugated to areporter group. Preferred reporter groups include enzymes (such ashorseradish peroxidase), substrates, cofactors, inhibitors, dyes,radionuclides, luminescent groups, fluorescent groups and biotin. Theconjugation of a binding agent to the reporter group may be achievedusing standard methods known to those of ordinary skill in the art.Common binding agents may also be purchased conjugated to a variety ofreporter groups from many commercial sources (e.g., Zymed Laboratories,San Francisco, Calif., and Pierce, Rockford, Ill.).

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound antibody. An appropriate amount of time may generally bedetermined from the manufacturer's instructions or by assaying the levelof binding that occurs over a period of time. Unbound detection reagentis then removed and bound detection reagent is detected using thereporter group. The method employed for detecting the reporter groupdepends upon the nature of the reporter group. For radioactive groups,scintillation counting or autoradiographic methods are generallyappropriate. Spectroscopic methods may be used to detect dyes,luminescent groups and fluorescent groups. Biotin may be detected usingavidin, coupled to a different reporter group (commonly a radioactive orfluorescent group or an enzyme). Enzyme reporter groups may generally bedetected by the addition of substrate (generally for a specific periodof time), followed by spectroscopic or other analysis of the reactionproducts.

To determine the presence or absence of anti-Mycobacterium antibodies inthe sample, the signal detected from the reporter group that remainsbound to the solid support is generally compared to a signal thatcorresponds to a predetermined cut-off value. In one preferredembodiment, the cut-off value is the average mean signal obtained whenthe immobilized antigen is incubated with samples from an uninfectedpatient. In general, a sample generating a signal that is three standarddeviations above the predetermined cut-off value is considered positivefor Mycobacterium infection. In another embodiment, the cut-off value isdetermined using a Receiver Operator Curve, according to the method ofSackett et al., Clinical Epidemiology: A Basic Science for ClinicalMedicine, Little Brown and Co., pp. 106-107 (1985). Briefly, in thisembodiment, the cut-off value may be determined from a plot of pairs oftrue positive rates (i.e., sensitivity) and false positive rates (100%specificity) that correspond to each possible cut-off value for thediagnostic test result. The cut-off value on the plot that is theclosest to the upper left-hand corner (i.e., the value that encloses thelargest area) is the most accurate cut-off value, and a samplegenerating a signal that is higher than the cut-off value determined bythis method may be considered positive. Alternatively, the cut-off valuemay be shifted to the left along the plot, to minimize the falsepositive rate, or to the right, to minimize the false negative rate. Ingeneral, a sample generating a signal that is higher than the cut-offvalue determined by this method is considered positive for tuberculosis.

In a related embodiment, the assay is performed in a rapid flow-throughor strip test format, wherein the antigen is immobilized on a membrane,such as, e.g., nitrocellulose. In the flow-through test, antibodieswithin the sample bind to the immobilized polypeptide as the samplepasses through the membrane. A detection reagent (e.g., proteinA-colloidal gold) then binds to the antibody-polypeptide complex as thesolution containing the detection reagent flows through the membrane.The detection of bound detection reagent may then be performed asdescribed above. In the strip test format, one end of the membrane towhich the polypeptide is bound is immersed in a solution containing thesample. The sample migrates along the membrane through a regioncontaining the detection reagent and to the area of immobilizedpolypeptide. The concentration of the detection reagent at thepolypeptide indicates the presence of anti-Mycobacterium antibodies inthe sample. Typically, the concentration of detection reagent at thatsite generates a pattern, such as a line, that can be read visually. Theabsence of such a pattern indicates a negative result. In general, theamount of polypeptide immobilized on the membrane is selected togenerate a visually discernible pattern when the biological samplecontains a level of antibodies that would be sufficient to generate apositive signal in an ELISA, as discussed supra. Preferably, the amountof polypeptide immobilized on the membrane ranges from about 25 ng toabout 1 μg, and more preferably from about 50 ng to about 500 ng. Suchtests can typically be performed with a very small amount (e.g., onedrop) of patient serum or blood.

In another aspect, this invention provides methods for using one or moreof the polypeptides described above to diagnose Mycobacterium infection,and in particular tuberculosis, using a skin test. As used herein, a“skin test” is any assay performed directly on a patient in which adelayed-type hypersensitivity (DTH) reaction (such as swelling,reddening or dermatitis) is measured following intradermal injection ofone or more polypeptides as described above. Such injection may beachieved using any suitable device sufficient to contact the polypeptideor polypeptides with dermal cells of the patient, such as a tuberculinsyringe or 1 ml syringe. Preferably, the reaction is measured at least48 hours after injection, more preferably 48-72 hours.

The DTH reaction is a cell-mediated immune response which is greater inpatients that have been exposed previously to the test antigen (i.e.,the immunogenic portion of the polypeptide employed, or a variantthereof). The response may be measured visually, using a ruler. Ingeneral, a response that is greater than about 0.5 cm in diameter,preferably greater than about 1.0 cm in diameter, is a positiveresponse, indicative of Mycobacterium infection, which may or may not bemanifested as an active disease.

The polypeptides of this invention are preferably formulated, for use ina skin test, as pharmaceutical compositions containing a polypeptide anda physiologically acceptable carrier, as described infra. Suchcompositions typically contain one or more of the above polypeptides inan amount ranging from about 1 μg to about 100 μg, preferably from about10 μg to about 50 μg in a volume of 0.1 ml. Preferably, the carrieremployed in such pharmaceutical compositions is a saline solution withappropriate preservatives, such as phenol and/or TWEEN 80™.

In a preferred embodiment, a polypeptide employed in a skin test is ofsufficient size such that it remains at the site of injection for theduration of the reaction period. In general, a polypeptide that is atleast 9 amino acids in length is sufficient. The polypeptide is alsopreferably broken down by macrophages within hours of injection to allowpresentation to T-cells. Such polypeptides may contain repeats of one ormore of the above sequences and/or other immunogenic or non-immunogenicsequences.

Of course, numerous other assay protocols exist that are suitable foruse with the polypeptides of the present invention. The abovedescriptions are intended to be exemplary only.

B. Diagnostic Assays with Polynucleotides Encoding MycobacteriumPolypeptides

Antibodies may be used in diagnostic tests to detect the presence ofMycobacterium antigens using assays similar to those detailed above andother techniques well known to those of skill in the art, therebyproviding a method for detecting Mycobacterium infection, and inparticular tuberculosis, in a patient.

Diagnostic reagents of the present invention may also comprise DNAsequences encoding one or more of the above polypeptides, or one or moreportions thereof. Alternatively, Mycobacterium infection can be detectedbased on the level of mRNA encoding a Mycobacterium antigen in abiological sample. For example, at least two oligonucleotide primers maybe employed in a polymerase chain reaction (PCR) based assay to amplifyMycobacterium-specific cDNA derived from a biological sample, wherein atleast one of the oligonucleotide primers is specific for (i.e.,hybridizes to) a DNA molecule encoding a polypeptide of the presentinvention. The presence of the amplified cDNA is then detected usingtechniques well known in the art, such as gel electrophoresis.Similarly, oligonucleotide probes specific for a DNA molecule encoding apolypeptide of the present invention may be used in a hybridizationassay to detect the presence of a polypeptide of the invention in abiological sample.

To permit hybridization under assay conditions, oligonucleotide primersand probes should comprise an oligonucleotide sequence that has at leastabout 60%, preferably at least about 75% and more preferably at leastabout 90%, identity to a portion of a polynucleotide encoding aMycobacterium antigen that is at least 10 nucleotides, and preferably atleast 20 nucleotides, in length. Preferably, oligonucleotide primersand/or probes hybridize to a polynucleotide encoding a polypeptidedescribed herein under moderately stringent conditions, as definedabove. Oligonucleotide primers and/or probes which may be usefullyemployed in the diagnostic methods described herein preferably are atleast 10-40 nucleotides in length. In a preferred embodiment, theoligonucleotide primers comprise at least 10 contiguous nucleotides,more preferably at least 15 contiguous nucleotides, of a DNA moleculehaving the sequence of SEQ ID NO:145, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 162, and 164. Primers or probes may thus beused to detect Mycobacterium-specific sequences in biological samples.DNA probes or primers comprising oligonucleotide sequences describedabove may be used alone, in combination with each other, or withpreviously identified sequences, such as the 38 kD antigen discussedabove.

Techniques for both PCR based assays and hybridization assays are wellknown in the art (see, for example, Mullis et al., Cold Spring HarborSymp. Quant. Biol., 51:263 (1987); Erlich ed., PCR Technology, StocktonPress, NY (1989)).

One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample and is reverse transcribed to produce cDNAmolecules. PCR amplification using at least one specific primergenerates a cDNA molecule, which may be separated and visualized using,for example, gel electrophoresis. Amplification may be performed onbiological samples taken from a test patient and from an individual whois not afflicted with Mycobacterium infection. The amplificationreaction may be performed on several dilutions of cDNA spanning twoorders of magnitude. A two-fold or greater increase in expression inseveral dilutions of the test patient sample as compared to the samedilutions of the non-infected sample is typically considered positive.

C. Diagnostic Assays Using the Detection of T Cells

A Mycobacterium infection may also, or alternatively, be detected basedon the presence of T cells that specifically react with a Mycobacteriumprotein in a biological sample. Within certain methods, a biologicalsample comprising CD4⁺ and/or CD8⁺ T cells isolated from a patient isincubated with a Mycobacterium polypeptide, a polynucleotide encodingsuch a polypeptide and/or an APC that expresses at least an immunogenicportion of such a polypeptide, and the presence or absence of specificactivation of the T cells is detected. Suitable biological samplesinclude, but are not limited to, isolated T cells. For example, T cellsmay be isolated from a patient by routine techniques (such as byFicoll/Hypaque density gradient centrifugation of peripheral bloodlymphocytes). T cells may be incubated in vitro for 2-9 days (typically4 days) at 37° C. with a Mycobacterium polypeptide of the invention (ata concentration of, e.g., 5-25 μg/ml). It may be desirable to incubateanother aliquot of a T cell sample in the absence of the Mycobacteriumpolypeptide to serve as a control. For CD4⁺ T cells, activation ispreferably detected by evaluating proliferation of the T cells. For CD8⁺T cells, activation is preferably detected by evaluating cytolyticactivity. A level of proliferation that is at least two fold greaterand/or a level of cytolytic activity that is at least 20% greater thanin disease-free patients indicates the presence of a Mycobacteriuminfection in the patient.

D. Diagnostic Assays for Monitoring the Progression of the Infection

In another embodiment, Mycobacterium proteins and polynucleotidesencoding such proteins may be used as markers for monitoring theprogression of a Mycobacterium infection. In this embodiment, assays asdescribed above for the diagnosis of a Mycobacterium infection may beperformed over time, and the change in the level of reactivepolypeptide(s) evaluated. For example, the assays may be performed every24-72 hours for a period of 1 month to 6-12 months, and thereafterperformed as needed. In general, the Mycobacterium infection isprogressing in those patients in whom the level of polypeptide detectedby the binding agent increases over time. In contrast, the Mycobacteriuminfection is not progressing when the level of reactive polypeptideeither remains constant or decreases with time.

As noted above, to improve sensitivity, multiple Mycobacterium markersmay be assayed within a given sample. It will be apparent that bindingagents specific for different proteins provided herein may be combinedwithin a single assay. Further, multiple primers or probes may be usedconcurrently. The selection of Mycobacterium protein markers may bebased on routine experiments to determine combinations that result inoptimal sensitivity.

VII. Therapeutic Applications

In another aspect, the present invention provides methods for using oneor more of the above polypeptides or fusion proteins (or DNA moleculesencoding such polypeptides) to induce protective immunity againstMycobacterium infection in a patient to either prevent or treatMycobacterium infection, and in particular tuberculosis.

A. Pharmaceutical Compositions

In additional embodiments, the present invention concerns formulation ofthe polypeptides, fusion proteins or DNA molecules disclosed herein inpharmaceutically-acceptable solutions for administration to a cell or ananimal, either alone, or in combination with one or more othermodalities of therapy. The pharmaceutical compositions of the inventionmay comprise one or more polypeptides, each of which may contain one ormore of the above sequences (or variants thereof), and a physiologicallyacceptable carrier.

It will also be understood that, if desired, the polypeptide, fusionprotein and nucleic acid molecule compositions disclosed herein may beadministered in combination with other agents as well, such as, e.g.,other proteins or polypeptides or various pharmaceutically-activeagents. In particular, such pharmaceutical compositions may also containother Mycobacterium antigens, either incorporated into a combinationpolypeptide or present within a separate polypeptide. In fact, there isvirtually no limit to other components that may also be included, giventhat the additional agents do not cause a significant adverse effectupon contact with the target cells or host tissues. The compositions maythus be delivered along with various other agents as required in theparticular instance. Such compositions may be purified from host cellsor other biological sources, or alternatively may be chemicallysynthesized as described herein. Likewise, such compositions may furthercomprise substituted or derivatized RNA or DNA compositions.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

1. Oral Administration

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (see, e.g.,Mathiowitz et al., Nature 386:410-414 (1997); Hwang et al., Crit RevTher Drug Carrier Syst. 15:243-84 (1998); U.S. Pat. Nos. 5,641,515;5,580,579; and 5,792,451). The tablets, troches, pills, capsules and thelike may also contain the following: a binder, as gum tragacanth,acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate;a disintegrating agent, such as corn starch, potato starch, alginic acidand the like; a lubricant, such as magnesium stearate; and a sweeteningagent, such as sucrose, lactose or saccharin may be added or a flavoringagent, such as peppermint, oil of wintergreen, or cherry flavoring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. A syrup of elixir may contain the activecompound sucrose as a sweetening agent methyl and propylparabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

2. Injectable Delivery

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally as describedin, e.g., U.S. Pat. Nos. 5,543,158; 5,641,515; and 5,399,363. Solutionsof the active compounds as free base or pharmacologically acceptablesalts may be prepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions may also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468). In all cases the form must besterile and must be fluid to the extent that easy syringability exists.It must be stable under the conditions of manufacture and storage andmust be preserved against the contaminating action of microorganisms,such as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington Pharmaceutical Sciences15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosagewill necessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,and the general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

3. Nasal Delivery

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212. Likewise, thedelivery of drugs using intranasal microparticle resins (Takenaga etal., J Controlled Release 52:81-87 (1998)) and lysophosphatidyl-glycerolcompounds (see, e.g., U.S. Pat. No. 5,725,871) are also well-known inthe pharmaceutical arts. Likewise, transmucosal drug delivery in theform of a polytetrafluoroetheylene support matrix is described in U.S.Pat. No. 5,780,045.

4. Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, or ananoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the polypeptides, fusionproteins and nucleic acids disclosed herein. The formation and use ofliposomes is generally known to those of skill in the art (see, e.g.,Couvreur et al., FEBS Lett. 84(2):323-326 (1977); Couvreur (1988);Lasic, Trends Biotechnol. 16(7):307-321 (1998); which describes the useof liposomes and nanocapsules in the targeted antibiotic therapy forintracellular bacterial infections and diseases). Recently, liposomeswere developed with improved serum stability and circulation half-times(Gabizon and Papahadjopoulos, Proc Natl Acad Sci USA. 85(18):6949-6953(1988); Allen and Choun (1987); U.S. Pat. No. 5,741,516). Further,various methods of liposome and liposome like preparations as potentialdrug carriers have been reviewed (Takakura, Nippon Rinsho 56(3):691-695(1998); Chandran et al., Indian J Exp Biol. 35(8):801-809 (1997);Margalit, Crit Rev Ther Drug Carrier Syst. 12(2-3):233-261 (1995); U.S.Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868; and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J Biol. Chem. 265(27):16337-16342 (1990); Muller et al., DNACell Biol. 9(3):221-229 (1990)). In addition, liposomes are free of theDNA length constraints that are typical of viral-based delivery systems.Liposomes have been used effectively to introduce genes, drugs (Heathand Martin, Chem Phys Lipids 40(2-4):347-358 (1986); Heath et al.,Biochim Biophys Acta. 862(1):72-80 (1986); Balazsovits et al., CancerChemother Pharmacol. 23(2):81-6. (1989); Fresta and Puglisi, J. DrugTarget 4(2):95-101 (1996)), radiotherapeutic agents (Pikul et al., ArchSurg. 122(12):1417-1420 (1987)), enzymes (Imaizumi et al., Stroke21(9):1312-1317 (1990); Imaizumi et al., Acta Neurochir Suppl (Wien)51:236-238 (1990)), viruses (Faller and Baltimore, J Virol.49(1):269-272 (1984)), transcription factors and allosteric effectors(Nicolau and Gersonde, Naturwissenschaften 66(11):563-566 (1979)) into avariety of cultured cell lines and animals. In addition, severalsuccessful clinical trails examining the effectiveness ofliposome-mediated drug delivery have been completed (Lopez-Berestein etal., J Infect Dis. 151(4):704-710 (1985); Lopez-Berestein et al., CancerDrug Deliv. 2(3):183-189 (1985); Coune, Infection 16(3):141-147 (1988);Sculier et al., Eur. J. Cancer Clin. Oncol. 24(3):527-38 (1988)).Furthermore, several studies suggest that the use of liposomes is notassociated with autoimmune responses, toxicity or gonadal localizationafter systemic delivery (Mori and Fukatsu, Epilepsia 33(6):994-1000(1992)).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplatedfor use in connection with the present invention as carriers for thepeptide compositions. They are widely suitable as both water- andlipid-soluble substances can be entrapped, i.e. in the aqueous spacesand within the bilayer itself, respectively. It is possible that thedrug-bearing liposomes may even be employed for site-specific deliveryof active agents by selectively modifying the liposomal formulation.

In addition to the teachings of Couvreur et al. (1977), supra; Couvreuret al. (1988), supra), the following information may be utilized ingenerating liposomal formulations. Phospholipids can form a variety ofstructures other than liposomes when dispersed in water, depending onthe molar ratio of lipid to water. At low ratios the liposome is thepreferred structure. The physical characteristics of liposomes depend onpH, ionic strength and the presence of divalent cations. Liposomes canshow low permeability to ionic and polar substances, but at elevatedtemperatures undergo a phase transition which markedly alters theirpermeability. The phase transition involves a change from a closelypacked, ordered structure, known as the gel state, to a loosely packed,less-ordered structure, known as the fluid state. This occurs at acharacteristic phase-transition temperature and results in an increasein permeability to ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins, such as cytochromec, bind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for antibiotic and inhibitordelivery will contain cholesterol.

The ability to trap solutes varies between different types of liposomes.For example, MLVs are moderately efficient at trapping solutes, but SUVsare extremely inefficient. SUVs offer the advantage of homogeneity andreproducibility in size distribution, however, and a compromise betweensize and trapping efficiency is offered by large unilamellar vesicles(LUVs). These are prepared by ether evaporation and are three to fourtimes more efficient at solute entrapment than MLVs.

In addition to liposome characteristics, an important determinant inentrapping compounds is the physicochemical properties of the compounditself. Polar compounds are trapped in the aqueous spaces and nonpolarcompounds bind to the lipid bilayer of the vesicle. Polar compounds arereleased through permeation or when the bilayer is broken, but nonpolarcompounds remain affiliated with the bilayer unless it is disrupted bytemperature or exposure to lipoproteins. Both types show maximum effluxrates at the phase transition temperature.

Liposomes interact with cells via four different mechanisms: endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity, and surface charge.They may persist in tissues for hours or days, depending on theircomposition, and half lives in the blood range from minutes to severalhours. Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. Antibodies may be used to bind tothe liposome surface and to direct the antibody and its drug contents tospecific antigenic receptors located on a particular cell-type surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

Alternatively, the invention provides for pharmaceutically-acceptablenanocapsule formulations of the compositions of the present invention.Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al. (1987); Quintanar-Guerrero et al., PharmRes. 15(7): 1056-1062 (1998); Douglas et al., Crit. Rev. Ther. DrugCarrier Syst. 3(3):233-261 (1987)). To avoid side effects due tointracellular polymeric overloading, such ultrafine particles (sizedaround 0.1 μm) should be designed using polymers able to be degraded invivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meetthese requirements are contemplated for use in the present invention.Such particles may be are easily made, as described (Couvreur et al., J.Pharm. Sci. 69(2): 199-202 (1980); Couvreur et al., (1988), supra; zurMuhlen et al., Eur. J. Pharm. Biopharm. 45(2): 149-155 (1998); Zambauxet al., J. Controlled Release 50(1-3):31-40 (1998); Pinto-Alphandry etal. (1995); and U.S. Pat. No. 5,145,684).

B. Vaccines

In certain preferred embodiments of the present invention, vaccines areprovided. The vaccines will generally comprise one or morepharmaceutical compositions, such as those discussed above, incombination with a non-specific immune response enhancer. A non-specificimmune response enhancer may be any substance that enhances orpotentiates an immune response (antibody and/or cell-mediated) to anexogenous antigen. Examples of non-specific immune response enhancersinclude adjuvants, biodegradable microspheres (e.g., polylacticgalactide) and liposomes (into which the compound is incorporated; seee.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccine preparation isgenerally described in, for example, Powell and Newman, eds., “VaccineDesign (the subunit and adjuvant approach),” Plenum Press (NY, 1995).Vaccines may be designed to generate antibody immunity and/or cellularimmunity such as that arising from CTL or CD4+ T cells.

Pharmaceutical compositions and vaccines within the scope of the presentinvention may also contain other compounds, which may be biologicallyactive or inactive. For example, one or more immunogenic portions ofother Mycobacterium antigens may be present, either incorporated into afusion polypeptide or as a separate compound, within the composition orvaccine. Polypeptides may, but need not, be conjugated to othermacromolecules as described, for example, within U.S. Pat. Nos.4,372,945 and 4,474,757. Pharmaceutical compositions and vaccines maygenerally be used for prophylactic and therapeutic purposes.

Illustrative vaccines may contain DNA encoding one or more of thepolypeptides as described above, such that the polypeptide is generatedin situ. Such a polynucleotide may comprise DNA, RNA, a modified nucleicacid or a DNA/RNA hybrid. As noted above, the nucleic acid may bepresent within any of a variety of delivery systems known to those ofordinary skill in the art, including nucleic acid expression systems,bacteria and viral expression systems. Numerous gene delivery techniquesare well known in the art, such as those described by Rolland, Crit.Rev. Therap. Drug Carrier Systems 15:143-198 (1998), and referencescited therein. Appropriate nucleic acid expression systems contain thenecessary DNA sequences for expression in the patient (such as asuitable promoter and terminating signal). Bacterial delivery systemsinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope. In apreferred embodiment, the DNA may be introduced using a viral expressionsystem (e.g., vaccinia or other pox virus, retrovirus, or adenovirus),which may involve the use of a non-pathogenic (defective), replicationcompetent virus. Suitable systems are disclosed, for example, inFisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321 (1989);Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103 (1989); Flexner et al.,Vaccine 8:17-21 (1990); U.S. Pat. Nos. 4,603,112; 4,769,330; and5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627 (1988);Rosenfeld et al., Science 252:431-434 (1991); Kolls et al., Proc. Natl.Acad. Sci. USA 91:215-219 (1994); Kass-Eisler et al., Proc. Natl. Acad.Sci. USA 90:11498-11502 (1993); Guzman et al., Circulation 88:2838-2848(1993); and Guzman et al., Cir. Res. 73:1202-1207 (1993). Techniques forincorporating DNA into such expression systems are well known to thoseof ordinary skill in the art. The DNA may also be “naked,” as described,for example, in Ulmer et al., Science 259:1745-1749 (1993) and reviewedby Cohen, Science 259:1691-1692 (1993). The uptake of naked DNA may beincreased by coating the DNA onto biodegradable beads, which areefficiently transported into the cells. It will be apparent that avaccine may comprise both a polynucleotide and a polypeptide component.Such vaccines may provide for an enhanced immune response.

In a related aspect, a DNA vaccine as described supra may beadministered simultaneously with or sequentially to either a polypeptideof the present invention or a known Mycobacterium antigen, such as the38 kD antigen described above For example, administration of DNAencoding a polypeptide of the present invention, either “naked” or in adelivery system as described supra, may be followed by administration ofan antigen in order to enhance the protective immune effect of thevaccine.

It will be apparent that a vaccine may contain pharmaceuticallyacceptable salts of the polynucleotides and polypeptides providedherein. Such salts may be prepared from pharmaceutically acceptablenon-toxic bases, including organic bases (e.g., salts of primary,secondary and tertiary amines and basic amino acids) and inorganic bases(e.g., sodium, potassium, lithium, ammonium, calcium and magnesiumsalts).

While any suitable carrier known to those of ordinary skill in the artmay be employed in the vaccine compositions of this invention, the typeof carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;5,814,344 and 5,942,252. One may also employ a carrier comprising theparticulate-protein complexes described in U.S. Pat. No. 5,928,647,which are capable of inducing a class I-restricted cytotoxic Tlymphocyte responses in a host.

Such compositions may also comprise buffers (e.g., neutral bufferedsaline or phosphate buffered saline), carbohydrates (e.g., glucose,mannose, sucrose or dextrans), mannitol, proteins, polypeptides or aminoacids such as glycine, antioxidants, bacteriostats, chelating agentssuch as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide),solutes that render the formulation isotonic, hypotonic or weaklyhypertonic with the blood of a recipient, suspending agents, thickeningagents and/or preservatives. Alternatively, compositions of the presentinvention may be formulated as a lyophilizate. Compounds may also beencapsulated within liposomes using well known technology.

Any of a variety of immunostimulants may be employed in the vaccines ofthis invention. For example, an adjuvant may be included. Most adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium species or Mycobacterium derived proteins. For example,delipidated, deglycolipidated M. vaccae (“pVac”) can be used. In anotherembodiment, BCG is used as an adjuvant. In addition, the vaccine can beadministered to a subject previously exposed to BCG. Suitable adjuvantsare commercially available as, for example, Freund's Incomplete Adjuvantand Complete Adjuvant (Difco Laboratories, Detroit, Mich.); MerckAdjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 andderivatives thereof (SmithKline Beecham, Philadelphia, Pa.); CWS, TDM,Leif, aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

Within the vaccines provided herein, the adjuvant composition ispreferably designed to induce an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann & Coffman, Ann. Rev.Immunol. 7:145-173 (1989).

Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), togetherwith an aluminum salt. MPL adjuvants are available from CorixaCorporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352 (1996). Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol^(R) toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 as disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

Other preferred adjuvants include Montanide ISA 720 (Seppic, France),SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), theSBAS series of adjuvants (e.g., SBAS-2, AS2′, AS2″, SBAS-4, or SBAS6,available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa,Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkylglucosaminide 4-phosphates (AGPs), such as those described in pendingU.S. patent application Ser. Nos. 08/853,826 and 09/074,720, thedisclosures of which are incorporated herein by reference in theirentireties, and polyoxyethylene ether adjuvants such as those describedin WO 99/52549A1.

Other preferred adjuvants include adjuvant molecules of the generalformula (I): HO(CH₂CH₂O)_(n)-A-R,

wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or PhenylC₁₋₅₀ alkyl.

One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

The polyoxyethylene ether according to the general formula (I) abovemay, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

Any vaccine provided herein may be prepared using well known methodsthat result in a combination of antigen, immune response enhancer and asuitable carrier or excipient. The compositions described herein may beadministered as part of a sustained release formulation (i.e., aformulation such as a capsule, sponge or gel (composed ofpolysaccharides, for example) that effects a slow release of compoundfollowing administration). Such formulations may generally be preparedusing well known technology (see, e.g., Coombes et al., Vaccine14:1429-1438 (1996)) and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.

Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. Such carriers includemicroparticles of poly(lactide-co-glycolide), polyacrylate, latex,starch, cellulose, dextran and the like. Other delayed-release carriersinclude supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638). The amount of active compound contained within a sustainedrelease formulation depends upon the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

C. Delivery Vehicles

Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of anantigen-specific immune response that targets infected cells. Deliveryvehicles include antigen presenting cells (APCs), such as dendriticcells, macrophages, B cells, monocytes and other cells that may beengineered to be efficient APCs. Such cells may, but need not, begenetically modified, e.g., to increase the capacity for presenting theantigen, to improve activation and/or maintenance of the T cell responseand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs and may be autologous, allogeneic,syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251 (1998)) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticimmunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529 (1999)). Ingeneral, dendritic cells may be identified based on their typical shape(stellate in situ, with marked cytoplasmic processes (dendrites) visiblein vitro), their ability to take up process and present antigens withhigh efficiency and their ability to activate naïve T cell responses.Dendritic cells may, of course, be engineered to express specificcell-surface receptors or ligands that are not commonly found ondendritic cells in vivo or ex vivo, and such modified dendritic cellsare contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600 (1998)).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, lymph nodes, spleen, skin, umbilical cord blood or anyother suitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce maturation and proliferation of dendriticcells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide encoding aMycobacterium antigen (or portion or other variant thereof) such thatthe Mycobacterium polypeptide, or an immunogenic portion thereof, isexpressed on the cell surface. Such transfection may take place ex vivo,and a composition or vaccine comprising such transfected cells may thenbe used for therapeutic purposes, as described herein. Alternatively, agene delivery vehicle that targets a dendritic or other antigenpresenting cell may be administered to a patient, resulting intransfection that occurs in vivo. In vivo and ex vivo transfection ofdendritic cells, for example, may generally be performed using anymethods known in the art, such as those described in, e.g., WO 97/24447,or the gene gun approach described by Mahvi et al., Immunology and cellBiology 75:456-460 (1997). Antigen loading of dendritic cells may beachieved by incubating dendritic cells or progenitor cells with theMycobacterium polypeptide, DNA (naked or within a plasmid vector) orRNA; or with antigen-expressing recombinant bacterium or viruses (e.g.,vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading,the polypeptide may be covalently conjugated to an immunological partnerthat provides T cell help (e.g., a carrier molecule). Alternatively, adendritic cell may be pulsed with a non-conjugated immunologicalpartner, separately or in the presence of the polypeptide.

D. Therapeutic Applications of the Compositions of the Invention

In further aspects of the present invention, the compositions describedsupra may be used for immunotherapy of Mycobacterium infection, and inparticular tuberculosis. Within such methods, pharmaceuticalcompositions and vaccines are typically administered to a patient toeither prevent the development of Mycobacterium infection or to treat apatient afflicted with Mycobacterium infection. Mycobacterium infectionmay be diagnosed using criteria generally accepted in the art, such as,e.g., in the case of tuberculosis, fever, acute inflammation of the lungand/or non-productive cough. Pharmaceutical compositions and vaccinesmay be administered either prior to or following a treatment such asadministration of conventional drugs. Administration may be by anysuitable route, including, e.g., intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal, intradermal, oral, etc.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against Mycobacterium infection with theadministration of immune response-modifying agents (such as polypeptidesand polynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedMycobacterium-immune reactivity (such as effector cells or antibodies)that can directly or indirectly mediate anti-Mycobacterium infectioneffects and do not necessarily depend on an intact host immune system.Examples of effector cells include T cells as discussed above, Tlymphocytes (such as CD8⁺ cytotoxic T lymphocytes and CD4⁺ T-helpertumor-infiltrating lymphocytes), killer cells (such as Natural Killercells and lymphokine-activated killer cells), B cells andantigen-presenting cells (such as dendritic cells and macrophages)expressing a polypeptide of the invention. T cell receptors and antibodyreceptors specific for the polypeptides recited herein may be cloned,expressed and transferred into other vectors or effector cells foradoptive immunotherapy. The polypeptides provided herein may also beused to generate antibodies or anti-idiotypic antibodies (as describedabove and in U.S. Pat. No. 4,918,164) for passive immunotherapy.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage or B cells, maybe pulsed with immunoreactive polypeptides or transfected with one ormore polynucleotides using standard techniques well known in the art.For example, antigen-presenting cells can be transfected with apolynucleotide having a promoter appropriate for increasing expressionin a recombinant virus or other expression system. Cultured effectorcells for use in therapy must be able to grow and distribute widely, andto survive long term in vivo. Studies have shown that cultured effectorcells can be induced to grow in vivo and to survive long term insubstantial numbers by repeated stimulation with antigen supplementedwith IL-2 (see, for example, Cheever et al., Immunological Reviews157:177, (1997)).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by, e.g., injection,intranasal or oral administration.

E. Formulation and Administration

Vaccines and pharmaceutical compositions may be presented in unit-doseor multi-dose containers, such as sealed ampoules or vials. Suchcontainers are preferably hermetically sealed to preserve sterility ofthe formulation until use. In general, formulations may be stored assuspensions, solutions or emulsions in oily or aqueous vehicles.Alternatively, a vaccine or pharmaceutical composition may be stored ina freeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

Routes and frequency of administration, as well as dosage, may vary fromindividual to individual and may parallel those currently being employedin immunization using BCG. In general, the pharmaceutical compositionsand vaccines may be administered, e.g., by injection (e.g.,intracutaneous, intramuscular, intravenous or subcutaneous),intranasally (e.g., by aspiration) or orally. Between 1 and 3 doses maybe administered for a 1-36 week period. Preferably, 3 doses areadministered, at intervals of 3-4 months, and booster vaccinations maybe given periodically thereafter. Alternate protocols may be appropriatefor individual patients. A suitable dose is an amount of polypeptide orDNA that, when administered as described supra, is capable of raising animmune response in an immunized patient sufficient to protect thepatient from Mycobacterium infection for at least 1-2 years. When usedfor a therapeutic purpose, a suitable dose is the amount that is capableof raising and immune response in a patient that is sufficient to obtainan improved clinical outcome (e.g., more frequent cure) in treatedpatients as compared to non-treated patients. Increases in preexistingimmune responses to a Mycobacterium protein generally correlate with animproved clinical outcome. Such immune responses may generally beevaluated using standard proliferation, cytotoxicity or cytokine assays,which may be performed using samples obtained from a patient before andafter treatment.

In general, the amount of polypeptide present in a dose (or produced insitu by the DNA in a dose) ranges from about 1 pg to about 100 mg per kgof host, typically from about 10 pg to about 1 mg, and preferably fromabout 100 pg to about 1 μg. Suitable dose sizes will vary with the sizeof the patient, but will typically range from about 0.1 ml to about 5ml.

F. Diagnostic Kits

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a Mycobacterium antigen. Suchantibodies or fragments may be provided attached to a support material,as described above. One or more additional containers may encloseelements, such as reagents or buffers, to be used in the assay. Suchkits may also, or alternatively, contain a detection reagent asdescribed above that contains a reporter group suitable for direct orindirect detection of antibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a Mycobacterium antigen in a biological sample. Such kitsgenerally comprise at least one oligonucleotide probe or primer, asdescribed above, that hybridizes to a polynucleotide encoding aMycobacterium antigen. Such an oligonucleotide may be used, for example,within a PCR or hybridization assay. Additional components that may bepresent within such kits include a second oligonucleotide and/or adiagnostic reagent or container to facilitate the detection of apolynucleotide encoding a Mycobacterium antigen.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

VIII. EXAMPLES Example 1 Purification and Characterization of M.Tuberculosis Polypeptides Using CD4+ T Cell Lines Generated from HumanPBMC

M. tuberculosis antigens of the present invention were isolated byexpression cloning of cDNA libraries of M. tuberculosis strains H37Rvand Erdman essentially as described by Sanderson et al. (J. Exp. Med.,182:1751-1757 (1995)) and were shown to induce PBMC proliferation andIFN-γ in an immunoreactive T cell line.

Two CD4+ T cell lines, referred to as DC-4 and DC-5, were generatedagainst dendritic cells infected with M. tuberculosis. Specifically,dendritic cells were prepared from adherent PBMC from a single donor andsubsequently infected with tuberculosis. Lymphocytes from the same donorwere cultured under limiting dilution conditions with the infecteddendritic cells to generate the CD4+ T cell lines DC-4 and DC-5. Thesecell lines were shown to react with crude soluble proteins from M.tuberculosis but not with Tb38-1. Limiting dilution conditions wereemployed to obtain a third CD4+ T cell line, referred to as DC-6, whichwas shown to react with both crude soluble proteins and Tb38-1.

Genomic DNA was isolated from the M. tuberculosis strains H37Rv andErdman and used to construct expression libraries in the vector pBSK(−)using the Lambda ZAP expression system (Stratagene, La Jolla, Calif.).These libraries were transformed into E. coli, pools of induced E. colicultures were incubated with dendritic cells, and the ability of theresulting incubated dendritic cells to stimulate cell proliferation andIFN-γ production in the CD4+ T cell line DC-6 was examined as describedbelow in Example 2. Positive pools were fractionated and re-tested untilpure M. tuberculosis clones were obtained.

Nineteen clones were isolated, of which nine were found to contain thepreviously identified M. tuberculosis antigens TbH-9 and Tb38-1,disclosed in U.S. patent application Ser. No. 08/533,634. The determinedcDNA sequences for the remaining ten clones (hereinafter referred to asTb224, Tb636, Tb424, Tb436, Tb398, Tb508, Tb441, Tb475, Tb488 and Tb465)are provided in SEQ ID NO:1-10, respectively. The correspondingpredicted amino acid sequences for Tb224 and Tb636 are provided in SEQID NO:13 and 14, respectively. The open reading frames for these twoantigens were found to show some homology to TbH-9. Tb224 and Tb636 werealso found to be overlapping clones.

Tb424, Tb436, Tb398. Tb508, Tb441, Tb475, Tb488 and Tb465 were eachfound to contain two small open reading frames (referred to as ORF-1 andORF-2) or truncated forms thereof, with minor variations in ORF-1 andORF-2 being found for each clone. The predicted amino acid sequences ofORF-1 and ORF-2 for Tb424, Tb436, Tb398, Tb508, Tb441, Tb475, Tb488 andTb465 are provided in SEQ ID NO:16 and 17, 18 and 19, 20 and 21, 22 and23, 24 and 25, 26 and 27, 28 and 29, and 30 and 31, respectively. Inaddition, clones Tb424 and Tb436 were found to contain a third apparentopen reading frame, referred to as ORF-U. The predicted amino acidsequences of ORF-U for Tb424 and Tb436 are provided in SEQ ID NO:32 and33, respectively. Tb424 and Tb436 were found to be either overlappingclones or recently duplicated/transposed copies. Similarly Tb398, Tb508and Tb465 were found to be either overlapping clones or recentlyduplicated/transposed copies, as were Tb475 and Tb488.

These sequences were compared with known sequences in publicly availablesequence databases using the BLASTN system. No homologies to theantigens Tb224 and Tb431 were found. Tb636 was found to be 100%identical to a cosmid previously identified in M. tuberculosis.Similarly, Tb508, Tb488, Tb398, Tb424, Tb436, Tb441, Tb465 and Tb475were found to show homology to known M. tuberculosis cosmids. Inaddition, Tb488 was found to have 100% homology to M. tuberculosistopoisomerase I.

Seventeen overlapping peptides to the open reading frames ORF-1(referred to as 1-1-1-17; SEQ ID NO:34-50, respectively) and thirtyoverlapping peptides to the open reading frame ORF-2 (referred to as2-1-2-30, SEQ ID NO:51-80, respectively) were synthesized using theprocedure described below in Example 4.

The ability of the synthetic peptides and of recombinant ORF-1 and ORF-2to induce T cell proliferation and IFN-γ production in PBMC fromPPD-positive donors was assayed as described below in Example 2. FIGS.1A-B and 2A-B illustrate stimulation of T cell proliferation and IFN-γby recombinant ORF-2 and the synthetic peptides 2-1-2-16 for two donors,referred to as D7 and D160, respectively. Recombinant ORF-2 (referred toas MTI) stimulated T cell proliferation and IFN-γ production in PBMCfrom both donors. The amount of PBMC stimulation seen with theindividual synthetic peptides varied with each donor, indicating thateach donor recognizes different epitopes on ORF-2. The proteins encodedby ORF-1, ORF-2 and ORF-U were subsequently named MTS, MTI and MSF,respectively.

Eighteen overlapping peptides to the sequence of MSF (referred to asMSF-1-MSF-18; SEQ ID NO:84-101, respectively) were synthesized and theirability to stimulate T cell proliferation and IFN-γ production in a CD4+T cell line generated against M. tuberculosis culture filtrate wasexamined as described below. The peptides referred to as MSF-12 andMSF-13 (SEQ ID NO:95 and 96, respectively) were found to show thehighest levels of reactivity.

Two overlapping peptides (SEQ ID NO:81 and 82) to the open reading frameof Tb224 were synthesized and shown to induce T cell proliferation andIFN-γ production in PBMC from PPD-positive donors.

Two CD4+ T cell lines from different donors were generated against M.tuberculosis infected dendritic cells using the above methodology.Screening of the M. tuberculosis cDNA expression library described aboveusing this cell line, resulted in the isolation of two clones referredto as Tb867 and Tb391. The determined cDNA sequence for Tb867 (SEQ IDNO:102) was found to be identical to the previously isolated M.tuberculosis cosmid SCY22G10, with the candidate reactive open readingframe encoding a 750 amino acid M. tuberculosis protein kinase.Comparison of the determined cDNA sequence for Tb391 (SEQ ID NO:103)with those in publicly available sequence databases revealed nosignificant homologies to known sequences.

In further studies, CD4+ T cell lines were generated against M.tuberculosis culture filtrate, essentially as outlined above, and usedto screen the M. tuberculosis Erdman cDNA expression library describedabove. Five reactive clones, referred to as Tb431, Tb472, Tb470, Tb838and Tb962 were isolated. The determined cDNA sequences for Tb431, Tb472,Tb470, and Tb838 are provided in SEQ ID NO:11, 12, 104 and 105,respectively, with the determined cDNA sequences for Tb962 beingprovided in SEQ ID NO:106 and 107. The corresponding predicted aminoacid sequence for Tb431 is provided in SEQ ID NO:15.

Subsequent studies led to the isolation of a full-length cDNA sequencefor Tb472 (SEQ ID NO:108). Overlapping peptides were synthesized andused to identify the reactive open reading frame. The predicted aminoacid sequence for the protein encoded by Tb472 (referred to as MSL) isprovided in SEQ ID NO:109. Comparison of the sequences for Tb472 and MSLwith those in publicly available sequence databases as described above,revealed no homologies to known sequences. Fifteen overlapping peptidesto the sequence of MSL (referred to as MSL-1-MSL-15; SEQ ID NO:110-124,respectively) were synthesized and their ability to stimulate T cellproliferation and IFN-γ production in a CD4+ T cell line generatedagainst M. tuberculosis culture filtrate was examined as describedbelow. The peptides referred to as MSL-10 (SEQ ID NO:119) and MSL-11(SEQ ID NO:120) were found to show the highest level of reactivity.Comparison of the determined cDNA sequence for Tb838 with those inpublicly available sequence databases revealed identity to thepreviously isolated M. tuberculosis cosmid SCY07H7. Comparison of thedetermined cDNA sequences for the clone Tb962 with those in publiclyavailable sequence databases revealed some homology to two previouslyidentified M. tuberculosis cosmids, one encoding a portion ofbactoferritin. However, recombinant bactoferritin was not found to bereactive with the T cell line used to isolate Tb962.

The clone Tb470, described above, was used to recover a full-length openreading frame (SEQ ID NO:125) that showed homology with TbH9 and wasfound to encode a 40 kDa antigen, referred to as Mtb40. The determinedamino acid sequence for Mtb40 is provided in SEQ ID NO:126. Similarly,subsequent studies led to the isolation of the full-length cDNA sequencefor Tb431, provided in SEQ ID NO:83, which was also determined tocontain an open reading frame encoding Mtb40. Tb470 and Tb431 were alsofound to contain a potential open reading frame encoding a U-ORF-likeantigen.

Screening of an M. tuberculosis Erdman cDNA expression library withmultiple CD4+ T cell lines generated against M. tuberculosis culturefiltrate, resulted in the isolation of three clones, referred to asTb366, Tb433 and Tb439. The determined cDNA sequences for Tb366, Tb433and Tb439 are provided in SEQ ID NO:127, 128 and 129, respectively.Comparison of these sequences with those in publicly available sequencedatabases revealed no significant homologies to Tb366. Tb433 was foundto show some homology to the previously identified M. tuberculosisantigen MPTS3. Tb439 was found to show 100% identity to the previouslyisolated M. tuberculosis cosmid SCY02B10.

A CD4+ T cell line was generated against M. tuberculosis PPD,essentially described above, and used to screen the above M.tuberculosis Erdman cDNA expression library. One reactive clone(referred to as Tb372) was isolated, with the determined cDNA sequencesbeing provided in SEQ ID NO:130 and 131. Comparison of these sequenceswith those in publicly available sequence databases revealed nosignificant homologies.

In further studies, screening of an M. tuberculosis cDNA expressionlibrary with a CD4+ T cell line generated against dendritic cells thathad been infected with tuberculosis for 8 days, as described above, ledto the isolation of two clones referred to as Th390R5C6 and Th390R2C11.The determined cDNA sequence for Tb390R5C6 is, provided in SEQ IDNO:132, with the determined cDNA sequences for Th390R2C 11 beingprovided in SEQ ID NO:133 and 134. Th390R5C6 was found to show 100%identity to a previously identified M. tuberculosis cosmid.

In subsequent studies, the methodology described above was used toscreen an M. tuberculosis genomic DNA library prepared as follows.Genomic DNA from M. tuberculosis Erdman strain was randomly sheared toan average size of 2 kb, and blunt ended with Klenow polymerase,followed by the addition of EcoRI adaptors. The insert was subsequentlyligated into the Screen phage vector (Novagen, Madison, Wis.) andpackaged in vitro using the PhageMaker extract (Novagen). The phagelibrary (referred to as the Erd λScreen library) was amplified and aportion was converted into a plasmid expression library by anautosubcloning mechanism using the E. coli strain BM25.8 (Novagen).Plasmid DNA was purified from BM25.8 cultures containing the pSCREENrecombinants and used to transform competent cells of the expressinghost strain BL21(DE3)pLysS. Transformed cells were aliquoted into 96well microtiter plates with each well containing a pool size ofapproximately 50 colonies. Replica plates of the 96 well plasmid libraryformat were induced with IPTG to allow recombinant protein expression.Following induction, the plates were centrifuged to pellet the E. coliwhich was used directly in T cell expression cloning of a CD4+ T cellline prepared from a PPD-positive donor (donor 160) as described above.Pools containing E. coli expressing M. tuberculosis T cell antigens weresubsequently broken down into individual colonies and reassayed in asimilar fashion to identify positive hits.

Screening of the T cell line from donor 160 with one 96 well plate ofthe Erd λScreen library provided a total of nine positive hits. Previousexperiments on the screening of the pBSK library described above with Tcells from donor 160 suggested that most or all of the positive cloneswould be TbH-9. Tb38-1 or MTI (disclosed in U.S. patent application Ser.No. 08/533,634) or variants thereof. However, Southern analysis revealedthat only three wells hybridized with a mixed probe of TbH-9, Tb38-1 andMTI. Of the remaining six positive wells, two were found to beidentical. The determined 5′ cDNA sequences for two of the isolatedclones (referred to as YI-26C1 and YI-86C11) are provided in SEQ IDNO:135 and 136, respectively. The full length cDNA sequence for theisolated clone referred to as hTcc#1 is provided in SEQ ID NO:137, withthe corresponding predicted amino acid sequence being provided in SEQ IDNO:138. Comparison of the sequences of hTcc#1 to those in publiclyavailable sequence databases as described above, revealed some homologyto the previously isolated M. tuberculosis cosmid MTCY07H7B.06.

Example 2 Induction of T Cell Proliferation and Interferon-γ Productionby M. tuberculosis Antigens

The ability of recombinant M. tuberculosis antigens to induce T-cellproliferation and interferon-γ production may be determined as follows.

Proteins may be induced by IPTG and purified by gel elution, asdescribed in Skeiky et al., J. Exp. Med. 181:1527-1537 (1995). Thepurified polypeptides are then screened for the ability to induce T-cellproliferation in PBMC preparations. The PBMCs from donors known to bePPD skin test positive and whose T-cells are known to proliferate inresponse to PPD are cultured in medium comprising RPMI 1640 supplementedwith 10% pooled human serum and 50 μg/ml gentamicin. Purifiedpolypeptides are added in duplicate at concentrations of 0.5 to 10μg/ml. After six days of culture in 96-well round-bottom plates in avolume of 200 μl, 50 μl of medium is removed from each well fordetermination of IFN-γ levels, as described below. The plates are thenpulsed with 1 μCi/well of tritiated thymidine for a further 18 hours,harvested and tritium uptake determined using a gas scintillationcounter. Fractions that result in proliferation in both replicates threefold greater than the proliferation observed in cells cultured in mediumalone are considered positive.

IFN-γ is measured using an enzyme-linked immunosorbent assay (ELISA).ELISA plates are coated with a mouse monoclonal antibody directed tohuman IFN-γ (PharMingen, San Diego, Calif.) in PBS for four hours atroom temperature. Wells are then blocked with PBS containing 5% (W/V)non-fat dried milk for 1 hour at room temperature. The plates are washedsix times in PBS/0.2% TWEEN-20 and samples diluted 1:2 in culture mediumin the ELISA plates are incubated overnight at room temperature. Theplates are again washed and a polyclonal rabbit anti-human IFN-γ serumdiluted 1:3000 in PBS/1 0% normal goat serum is added to each well. Theplates are then incubated for two hours at room temperature, washed andhorseradish peroxidase-coupled anti-rabbit IgG (Sigma Chemical So., St.Louis, Mo.) is added at a 1:2000 dilution in PBS/5% non-fat dried milk.After a further two hour incubation at room temperature, the plates arewashed and TMB substrate added. The reaction is stopped after 20 minwith 1 N sulfuric acid. Optical density is determined at 450 nm using570 nm as a reference wavelength. Fractions that result in bothreplicates giving an OD two fold greater than the mean OD from cellscultured in medium alone, plus 3 standard deviations, are consideredpositive.

Example 3 Purification and Characterization of M. tuberculosisPolypeptides Using CD4+ T Cell Lines Generated from a Mouse M.tuberculosis Model

Infection of C57BL/6 mice with M. tuberculosis results in thedevelopment of a progressive disease for approximately 2-3 weeks. Thedisease progression is then halted as a consequence of the emergence ofa strong protective T cell-mediated immune response. This infectionmodel was used to generate T cell lines capable of recognizingprotective M. tuberculosis antigens.

Specifically, spleen cells were obtained from C57BL/6 mice infected withM. tuberculosis for 28 days and used to raise specific anti-M.tuberculosis T cell lines as described above. The resulting CD4+ T celllines, in conjunction with normal antigen presenting (spleen) cells fromC57BL/6 mice were used to screen the M. tuberculosis Erd λScreen librarydescribed above. One of the reactive library pools, which was found tobe highly stimulatory of the T cells, was selected and the correspondingactive clone (referred to as Y288C10) was isolated.

Sequencing of the clone Y2SSC10 revealed that it contains two potentialgenes, in tandem. The determined cDNA sequences for these two genes(referred to as mTCC#1 and mTCC#2) are provided in SEQ ID NO:139 and140, respectively, with the corresponding predicted amino acid sequencesbeing provided in SEQ ID NO:141 and 142, respectively. Comparison ofthese sequences with those in publicly available sequence databasesrevealed identity to unknown sequences previously found within the M.tuberculosis cosmid MTY21C12. The predicted amino acid sequences ofmTCC#1 and mTCC#2 were found to show some homology to previouslyidentified members of the TbH9 protein family, discussed above.

Example 4 Synthesis of Synthetic Polypeptides

Polypeptides may be synthesized on a Millipore 9050 peptide synthesizerusing FMOC chemistry with HIPTU(O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence may be attached to the amino terminusof the peptide to provide a method of conjugation or labeling of thepeptide. Cleavage of the peptides from the solid support may be carriedout using the following cleavage mixture: trifluoroaceticacid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavingfor 2 hours, the peptides may be precipitated in coldmethyl-t-butyl-ether. The peptide pellets may then be dissolved in watercontaining 0.1% trifluoroacetic acid (TFA) and lyophilized prior topurification by C18 reverse phase HPLC. A gradient of 0-60% acetonitrile(containing 0.1% TFA) in water (containing 0.1% TFA) may be used toelute the peptides. Following lyophilization of the pure fractions, thepeptides may be characterized using electrospray mass spectrometry andby amino acid analysis.

Example 5 Use of Representative Antigens for Serodiagnosis ofTuberculosis

The diagnostic properties of representative M. tuberculosis antigens maybe determined by examining the reactivity of antigens with sera fromtuberculosis-infected patients and from normal donors as describedbelow.

Assays are performed in 96-well plates coated with 200 ng antigendiluted to 50 μl in carbonate coating buffer, pH 9.6. The wells arecoated overnight at 4° C. (or 2 hours at 37° C.). The plate contents arethen removed and the wells are blocked for 2 hours with 200 μl of PBS/1%BSA. After the blocking step, the wells are washed five times withPBS/0.1% Tween 20™. 50 μl sera, diluted 1:100 in PBS/0.1% Tween 20/0.1%BSA, is then added to each well and incubated for 30 minutes at roomtemperature. The plates are washed again five times with PBS/0.1% Tween20™.

The enzyme conjugate (horseradish peroxidase—Protein A, Zymed, SanFrancisco, Calif.) is then 1:10,000 in PBS/0.1% Tween20™/0.1% BSA, and50 μl of the diluted conjugate is added to each well and incubated for30 minutes at room temperature. Following incubation, the wells arewashed five times with PBS/0.1% Tween 20™. 100 μl oftetramethylbenzidine peroxidase (TMB) substrate (Kirkegaard and PerryLaboratories, Gaithersburg, Md.) is added, undiluted, and incubated forabout 15 minutes. The reaction is stopped with the addition of 100 μl of1 NH₂SO₄ to each well, and the plates are read at 450 nm.

Example 6 Murine T Cell Expression Cloning of an Mtb Antigen Associatedwith the Control of TB Infection

Genomic DNA form M. tuberculosis Erdman strain was randomly sheared toan average size of 2 kb, blunt ended with Klenow polymerase and followedby the addition of EcoRI adaptors. The insert was subsequently ligatedinto the Screen phage vector predigested with EcoRI (Novagen, Madison,Wis.) and packaged in vitro using the PhageMaker extract (Novagen,Madison, Wis.). The phage library (Erd Screen) was amplified and aportion converted into a plasmid expression library (pScreen) byautosubcloning using the E. coli host strain BM25.8 as suggested by themanufacturer (Novagen, Madison, Wis.). Plasmid DNA was purified fromBM25.8 cultures containing pScreen recombinants and used to transformcompetent cells of the expressing host strain BL21 (DE3)pLysS.Transformed cells were aliquoted into 96 well micro titer plates witheach well containing a pool size of ˜50 colonies. Replica plates of the96 well plasmid library format were induced with IPTG to allowrecombinant protein expression. Following induction, the plates werecentrifuged to pellet the E. coli and the bacterial pellet wasresuspended in 200 μl of 1×PBS. The general principle is based on thedirect recognition by the T cells of the antigens presented by antigenpresenting cells that have internalized a library of E. coli-containingexpressed recombinant antigens. The M. tuberculosis library wasinitially divided in pools containing approximately 50-100transformants/ml distributed in 96-well microtiter plates and stored ina replica plate manner. Adherent spleen cells were fed with the E. colipools and incubated for processing for 2 h. After washing the adherentcells were exposed to specific T cell lines in the presence ofgentamycin (50 μg/ml) to inhibit the bacterial growth. T cellrecognition of pool containing M. tuberculosis antigens was thendetected by proliferation (3H thymidine incorporation). Wells thatscored positive were then broken down using the same protocol until asingle clone was detected. The gene was then sequenced, sub-cloned,expressed and the recombinant protein evaluated. Nucleotide sequencecomparison of the 0.6 kb insert of clone mTTC#3 with the GenBankdatabase revealed that it is comprised of the amino terminal portion ofgene MTV014.03c (locus MTV014; accession # e1248750) of the Mtb H37Rvstrain. The full length nucleotide sequence of mTTC#3 (SEQ ID NO:145) isa 1.86 kb fragment comprising the entire ORF with a predicted molecularweight of ˜57 kDa (SEQ ID NO:146). Thus, to maintain consistency withour nomenclature, mTTC#3 is referred to hereafter as MTB57. The fulllength coding portion of mTTC#3 (MTB57) was PCR amplified using thefollowing primer pairs: 5′(5′-CAA TTA CAT ATG CAT CAC CAT CAC CAT CACATG AAT TAT TCG GTG TTG CCG (SEQ ID NO:147)) and 3′ (5′-CAA TTA AAG CTTTTA GGG CTG ACC GAA GAA GCC (SEQ ID NO:148))h3. The full length nucleicacid coding sequence of mTTC#3 and the corresponding predicted aminoacid sequence are provided in FIGS. 3 and 4, respectively.

Example 7 Identification of Mycobacterium Tuberculosis Antigens Excretedin Urine of Infected Mice

Antigen were prepared by infecting intravenously C57BL/6 mice with 4.10⁷colony forming units (CFU) of M. tuberculosis. 14 days later the animalswere bled and their urine was collected in microfuge tubes. Sera wereobtained at room temperature. Both sera and urine were centrifuged at10,000 g for 15 minutes followed by filtration in 0.2 u sterilemembranes.

Antibodies were produced against the antigens by immunizing normalC57BL/6 mice with either the sera or the urine from the M. tuberculosisinfected C57BL/6 mice. The adjuvant used was incomplete Freund'sadjuvant (IFA). Immunization was carried out according to the followingprotocol: on day 1, mice were injected in the footpad or in the base ofthe tail with a mix containing 100 μl of either serum or urine and 100μl of IFA; on day 14, a mix containing 100 μl of either serum or urineand 100 μl of IFA was injected intraperitoneally to the mice; finally onday 28, either 200 μl of serum or 50 μl of urine were injected to themice intraperitoneally. By using syngeneic mice for the antibodyproduction, only antibodies specific for foreign antigens present in theblood circulation or urine of the C57BL/6 mice, i.e., M. tuberculosisantigens, are generated. On day 35, 100 μl of blood were collected byeye-bleeding the immunized mice. ELISA assays were performed with theobtained sera using a M. tuberculosis crude lysate. The ELISAexperiments revealed that all the mice immunized with either sera orurine from infected donors produced anti-M. tuberculosisantibodies intiters varying from 1/40 to 1/320. No anti-M. tuberculosis antibodieswere found in the sera obtained from the mice before the immunizations.

The antiserum made against the proteins excreted in the urine was usedto screen a Mtb expression library prepared in the lambda screen phageexpression system. Positive clones were purified and their correspondinginserts sequenced. These inserts were named P1, 2, 3, 4, 6, 7, 8, 9, 10,11 and 12 (SEQ ID NO:149-159).

Example 8 Identification of Mycobacterium Tuberculosis Antigens UsingCD4+ T Cell Expression Cloning

Expression screening using a number of T cell lines generated fromhealthy PPD-positive individuals has been employed to identify M.tuberculosis clones encoding reactive antigens. Pools of M. tuberculosisrecombinant clones (expressed in E. coli) were fed to dendritic cells.Autologous T cell lines were incubated with the dendritic cells andproliferation and INF-gamma production was measured. Reactive pools werefractionated and re-tested until pure M. tuberculosis clones wereachieved. This approach allows for direct screening for T cell antigens.A related approach has been used to identify Listeria monocytogenesantigens (see J. Exp. Med. 182:1751-1757 (1995).

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention.

1-51. (canceled)
 52. An isolated polypeptide comprising an immunogenicportion of an M. tuberculosis antigen, wherein the antigen has at least90% amino acid sequence identity to SEQ ID NO:109.
 53. The polypeptideof claim 52, wherein the antigen has at least 95% amino acid sequenceidentity to SEQ ID NO:109.
 54. The polypeptide of claim 52, wherein theantigen consists of the amino acid sequence of SEQ ID NO:109.
 55. Thepolypeptide of claim 52, which comprises the amino acid sequence of SEQID NO:109.
 56. The polypeptide of claim 52, which consists of the aminoacid sequence of SEQ ID NO:109.
 57. The polypeptide of claim 52, whichcomprises the amino acid sequence of any one of SEQ ID NOs:110-124. 58.The polypeptide of claim 57, which comprises the amino acid sequence ofSEQ ID NO:119.
 59. The polypeptide of claim 58, which consists of theamino acid sequence of SEQ ID NO:119.
 60. The polypeptide of claim 57,which comprises the amino acid sequence of SEQ ID NO:120.
 61. Thepolypeptide of claim 60, which consists of the amino acid sequence ofSEQ ID NO:120.
 62. An isolated nucleic acid comprising a polynucleotidesequence encoding the polypeptide of claim
 52. 63. The nucleic acid ofclaim 62, which comprises the polynucleotide sequence of SEQ ID NO:108.64. The nucleic acid of claim 63, which consists of the polynucleotidesequence of SEQ ID NO:108.
 65. An expression vector comprising thenucleic acid of claim
 62. 66. An isolated host cell transformed with theexpression vector of claim
 65. 67. The host cell of claim 66, which isselected from the group consisting of E. coli, yeast, and mammaliancells.
 68. A pharmaceutical composition comprising the polypeptide ofclaim 52 and a physiologically acceptable carrier.
 69. A pharmaceuticalcomposition comprising the nucleic acid of claim 62 and aphysiologically acceptable carrier.
 70. A vaccine for inducingprotective immunity comprising the polypeptide of claim 52 and anon-specific immune response enhancer.
 71. The vaccine of claim 70,wherein the non-specific immune response enhancer is an adjuvant.
 72. Avaccine for inducing protective immunity comprising the nucleic acid ofclaim 62 and a non-specific immune response enhancer.
 73. The vaccine ofclaim 72, wherein the non-specific immune response enhancer is anadjuvant.
 74. A fusion protein comprising two polypeptides of claim 52.75. A fusion protein comprising the polypeptide of claim 52 and a secondM. tuberculosis antigen.
 76. A pharmaceutical composition comprising thefusion protein of claim 74 and a physiologically acceptable carrier. 77.A pharmaceutical composition comprising the fusion protein of claim 75and a physiologically acceptable carrier.
 78. A vaccine for inducingprotective immunity comprising the fusion protein of claim 74 and anon-specific immune response enhancer.
 79. A vaccine for inducingprotective immunity comprising the fusion protein of claim 75 and anon-specific immune response enhancer.
 80. The vaccine of claim 78 or79, wherein the non-specific immune response enhancer is an adjuvant.81. A diagnostic kit for detecting tuberculosis comprising: (a) thepolypeptide of claim 52; and (b) apparatus sufficient to contact saidpolypeptide with the dermal cells of a patient in order to induce immuneresponse on the patient's skin.
 82. The kit of claim 81, wherein theimmune response is induration.
 83. A method for inducing protectiveimmunity in a patient comprising administering the pharmaceuticalcomposition of any one of claims 68, 69, 76, and
 77. 84. A method forinducing protective immunity in a patient comprising administering thevaccine of any one of claims 70, 72, 78, and
 79. 85. A method fordetecting tuberculosis in a patient comprising: (a) contacting dermalcells of a patient with the polypeptide of claim 52; and (b) detectingan immune response on the patient's skin and therefrom detectingtuberculosis in the patient.